Journal of Integrative Agriculture
Advanced Online Publication 2015
Doi: 10.1016/S2095-3119(15)61192-3
A plastidic ATP/ADP transporter gene, IbAATP, increases starch and amylose content
and alters starch structure in transgenic sweetpotato
WANG Yan-nan, LI Yan, ZHANG Huan, ZHAI Hong, LIU Qing-chang, HE Shao-zhen*
China Agricultural University, College of Agronomy & Biotechnology, Beijing 100193, P.R.China
Abstract A plastidic ATP/ADP transporter (AATP) is responsible for importing ATP from the cytosol into
plastids. In dicotyledonous plants, increasing the ATP supply is a potential way to facilitate anabolic synthesis in
heterotrophic plastids. In this study, a gene encoding the AATP protein, named IbAATP, was isolated from
sweetpotato (Ipomoea batatas (L.) Lam.). Transcripts of IbAATP were predominantly detected in the storage
roots and leaves and were induced by exogenous sucrose and subject to circadian rhythm. Transient expression
of IbAATP in tobacco and onion epidermal cells revealed the plastidic localization of IbAATP. The
overexpression of IbAATP in sweetpotato significantly increased the starch and amylose content and led to
enlarged starch granules. The IbAATP-overexpressing plants showed altered fine structure of amylopectin,
which contained an increased proportion of chains with a degree of polymerization (DP) of 10-23 and a reduced
number of chains with a DP of 5-9 and 24-40. In addition, starch from the transgenic plants exhibited different
pasting properties. The transcript levels of starch biosynthetic genes, including IbAGP, IbGBSSI, IbSSI-IV and
IbSBE, were differentially regulated in the transgenic plants. These results revealed the explicit role of IbAATP
in the starch biosynthesis of sweetpotato and indicated that this gene has the potential to be used to improve starch
content and quality in sweetpotato and other plants.
Keywords: sweetpotato, IbAATP, starch content and composition, starch granule size, starch structure, pasting
properties
1. Introduction
Sweetpotato is considered to be the seventh most important food crop in the world (Food and Agricultural
Organization (FAO), 2009, FAO Statistics, http://faostat3.fao.org.). It is a staple food source for some
undeveloped regions and is considered a promising source of starch that can be processed into bioethanol
(Bovell-Benjamin 2007). In the past few years, the demand for liquid biofuel produced by the fermentation of
starch or sugars has increased considerably. However, starch or sugar can contribute only a small fraction to
biofuel production unless more land is used to develop this new industry (Smith 2008). Thus, the improvement
of the starch content and quality of sweetpotato is an urgent need, especially in the field of biotechnology.
Our knowledge of the enhancement of the starch content in sweetpotato through genetic manipulation is limited.
Tanaka et al. (2009) reported that overexpression of the IbSRF1 (a Dof zinc finger transcription factor) in
sweetpotato increased the starch content in the storage roots. In another report, Arabidopsis overexpressing the
sweetpotato IbEXP1 accumulated more starch and storage reserves in the seeds (Bae et al. 2014). However,
whether the overexpression of IbEXP1 in sweetpotato can alter the starch content in storage roots remains
unknown. Manipulation of the genes in the starch biosynthesis pathway (such as IbGBSSI, IbSBEII and IbSSII)
has an impact on the starch composition, structure and physicochemical properties but little influence on the
starch content in sweetpotato (Kimura et al. 2001; Kitahara et al. 2007; Takahata et al. 2010; Kitahara et al.
2011).
In below-ground storage organs, the best option to increase the starch content may lie in increasing the ATP
supply to the plastid (Smith 2008). ATP is the basic energy currency in living cells and is needed for almost
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every step of biochemical reactions. ATP is also an indispensable participant in the ADP-glucose
pyrophosphorylase (AGPase) reaction, which catalyzes the formation of ADP-glucose (ADP-Glc) and is
considered as a rate-limiting enzyme in starch biosynthesis (Jeon et al. 2010). ATP exchange between organelles
and the cytosol is mediated by adenylate carrier proteins. One type of adenylate carrier protein is the
mitochondrial ADP/ATP carrier (AAC), which can export ATP produced previously via oxidative phosphorylation
in a one to one exchange of cytosolic ADP (Fiore et al. 1998). The mitochondrial AAC is a dimer, and each
monomer contains 6 transmembrane helices (Winkler and Neuhaus 1999). Another type of adenylate carrier
protein is the plastidic ATP/ADP transporter protein (AATP), which was discovered in spinach chloroplasts.
AATP is generally found in the heterotrophic plastids (amyloplasts, chromoplasts and leucoplasts) of higher and
lower land plants as an important energy transporter (Heldt 1969; Schünemann et al. 1993; Emes and Neuhaus
1997; Möhlmann et al. 1998; Linka et al. 2003; Meng et al. 2005; Yuen et al. 2009). The main function of AATP
is to provide the plastid stroma with cytosolic ATP, which is required for anabolic processes such as starch and
fatty acid synthesis (Möhlmann et al. 1998).
A few studies have elucidated the function of AATP in starch biosynthesis in plants (Tjaden et al. 1998;
Geigenberger et al. 2001). In potato tubers, overexpression of the Arabidopsis AATP1 increased the level of
ADP-glucose up to 2-fold and the starch content by 16-36%. In contrast, the antisense inhibition of the potato
AATP1 reduced the level of ADP-glucose by 25-70% and the starch content by 19-51% (Tjaden et al. 1998;
Geigenberger et al. 2001). In another study, down-regulation of the activity of plastidic adenylate kinase, which
interconverts ATP and AMP into ADP, caused a substantial effect on the adenylate pool size and the ADP-glucose
level in potato tubers, further increasing the starch content (Regierer et al. 2002). These results indicate that the
manipulation of the enzymes that control the ATP supply in plastids is an effective way to enhance starch
biosynthesis in storage organs.
In sweetpotato, no study reported the impact of increasing the ATP supply or the role of AATP in starch
biosynthesis. In the present study, we isolated a plastidic ATP/ADP transporter gene (IbAATP) from sweetpotato.
Transgenic sweetpotato overexpressing IbAATP had an altered starch content, composition, starch granule size
and structure. The mechanism underlying these alterations is discussed.
2. Results
2.1. Cloning and sequence analysis of IbAATP
The IbAATP was cloned from the high-starch sweetpotato line Xu 781 using the method of rapid amplification of
cDNA ends (RACE). The cloned 2257-bp full-length IbAATP cDNA contained an 1890-bp ORF that produced a
polypeptide with a molecular weight of 68.3 kDa and a theoretical isoelectric point (pI) of 9.56. A search for the
amino acid sequence of IbAATP in NCBI returned a putative domain of the ADP/ATP carrier protein. The
genomic sequence of IbAATP was 3416 bp long and contained 5 exons and 4 introns. This genomic organization
showed similarity to the SlAATP from tomato (Solanum lycopersicum) and the StAATP1 from potato (Solanum
tuberosum). According to the online software TMHMM (v. 2.0), IbAATP contains 10 transmembrane (TM)
helices, which is one of the main structural characteristics of the AATP family (Winkler and Neuhaus 1999; Yuen
et al. 2009).
The deduced amino acid sequence alignment showed that IbAATP was highly conserved and homologous to the
AATPs from Solanum lycopersicum (SlAATP), Solanum tuberosum (StAATP1), Arabidopsis thaliana (AtAATP1;
2), Zea mays (ZmAATP), Oryza sativa (OsAATP1; 2) and Rickettsia prowazekii (TLCRp), especially in the
transmembrane (TM) regions (Fig. 1). However, the N-terminus varies among the AATPs from different species.
Homology analysis using DNAMAN indicated that IbAATP has 84.41 and 84.12% identity with SlAATP and
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StAATP1, respectively, in the amino acid sequence. Five highly conserved motifs (FLKT, AELWG, FANQIT,
AYG(I/V)S(I/V)NLVE, and (L/I)GKSGGA(L/I)IQ) present in the plant and bacterial ATP/ADP transporter
proteins were also identified, indicating the importance of these motifs in determining AATP function (Möhlmann
et al. 1998; Meng et al. 2005). A conserved alanine-rich N-terminal motif harboring the transit peptide (TP)
cleavage site in dicot members of AATPs was detected (Ile-Cys-(Arg/Lys)-Ala-Glu-Ala-Ala-Ala-Ala), whose first
amino acid was replaced by valine (Val) in IbAATP (Yuen et al. 2009). The predicted chloroplastic transit
peptide of IbAATP was 80 residues starting from methionine (Met) in the N-terminal extension, and the cleavage
site fell within this motif (Val-Cys-Arg↓Ala-Glu-Ala-Ala-Ala-Ala). Phylogenetic analysis further revealed that
IbAATP had a closer relationship with SlAATP from Solanum lycopersicum (84.41% homology in amino acid
sequence) and StAATP1 from Solanum tuberosum (84.12% homology in amino acid sequence) (Fig. 2). The
IbAATP was categorized into the dicot group which diverged from AATPs of monocots (Oryza sativa and Zea
mays).
2.2. Expression analysis of IbAATP in sweetpotato
The spatial expression pattern of the IbAATP was investigated using quantitative real-time PCR (qRT-PCR). The
results showed that the IbAATP transcript accumulated in all five of the major tissues in sweetpotato (leaf, stem,
petiole, storage root and fibrous root). The amount of transcript was significantly higher in the storage root and
leaf than in the stem, petiole and fibrous root (Fig. 3-A). This expression pattern was similar to that of AtNTT1
from Arabidopsis and MeAATP1/2 from cassava (Manihot esculenta Crantz) (Reiser et al. 2004; Yuen et al. 2009).
To examine whether the expression of IbAATP responded to exogenous sucrose, we floated young leaf-petioles
in water (control) or 175 mmol L-1 sucrose for up to 48 h. The results showed that no induction of the IbAATP
transcript occurred when the leaf-petiole cuttings were treated with water (Fig. 3-B). In contrast, the presence of
exogenous sucrose significantly increased the accumulation of the IbAATP transcript, reaching a peak at 12 h and
declining thereafter. The transcript level of IbAATP was 0.5-fold higher than that of the 0 h at the end of
treatment.
The circadian rhythm of plants is endogenously sustained in a cyclical manner with a free-running period of
approximately 24 h, which is especially frequent for genes assigned to starch, sucrose and trehalose metabolism
(Hillman 1976; Bläsing et al. 2005). As shown in Fig. 3-C, the transcript level of IbAATP oscillated during a
24-h day-night alteration (P1 to P4), and the transcript level at P5 was similar with that at P1 and then dropped to
a level (P6) that was lower than that at P2. This transcript curve followed a circadian oscillation as described by
Green et al. (2002), which indicated that the external light supply did not affect the transcript level of IbAATP
after the plants had acclimated to a 16-h light/8-h dark regimen. Similar to IbAATP, the transcript level of
IbGBSSI (positive control) displayed a sine-wave pattern. The differences in the transcript levels of IbAATP and
IbGBSSI between P5 and P1 and between P6 and P2 may have been due to variations in the individual growth
status of the treated in vitro-grown plants. These results demonstrated that IbAATP was regulated by the
circadian rhythm.
2.3. Subcellular localization of the IbAATP
Computer programs, such as TargetP and ChloroP, suggested a chloroplastic localization of IbAATP. An
IbAATP-green fluorescent protein (GFP) construct was therefore transiently expressed in tobacco (N.
benthamiana) epidermal cells and onion (A. cepa) epidermal cells and visualized using a laser scanning confocal
microscope (Nikon Inc., Melville, NY) (Fig. 4). In the tobacco epidermal cells, IbAATP was observed as
scattered patches and co-localized with the autofluorescence of chloroplasts. Similarly, the signal of the
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IbAATP-GFP fusion protein was dispersed in the onion peel epidermal cells as small spots and clava, which were
assumed to be the proplastids. These results are in good agreement with the plastidic localization of IbAATP.
2.4. Production of transgenic sweetpotato plants
To functionally characterize IbAATP, an overexpression vector (pC3301-121-IbAATP) was constructed and
introduced into cell aggregates of Lizixiang (Appendix A). A total of 64 phosphinothricin (PPT)-resistant
calluses were generated from approximately 1450 transfected cell aggregates after 8 wk of selection on Murashige
and Skoog (MS) medium with 2.0 mg L-1 2,4-D, 300 mg L-1 cefotaxime sodium and 50 mg L-1 PPT (Appendix A).
A total of 51 of these calluses formed 259 putative transgenic plants, named L1, L2, … , L259, after being
transferred to MS medium with 1.0 mg L-1 ABA and 300 mg L-1 cefotaxime sodium. Of the 259 putative
transgenic plants, 17 were proved to be transgenic by both the GUS staining assay and PCR analysis (Appendix
A). These 17 lines were planted in the soil in a greenhouse and then in the field (Appendix A). Storage roots
were harvested after 5 months growth in the field (Appendix A). The expression levels of mRNA for IbAATP in
the storage roots of the 17 transgenic lines were investigated using qRT-PCR (Fig. 5). Compared with the wild
type (WT), three lines (L151, L224 and L259) showed the highest IbAATP transcript level. These three lines
were selected for further phenotypic analysis.
2.5. Starch content and composition
The starch and amylose content in the storage roots of the three transgenic lines and the WT were quantified. All
three transgenic lines exhibited an increased starch level (Table 1). In L151, the starch level was increased by
57.3% compared with the WT (121.17 + 1.76 mg g-1 FW). In L224 and L259, increases in starch of 26.7 and
36.9%, respectively, were observed. Additionally, the overexpression of IbAATP caused a significant increase in
the amylose content in transgenic plants (Table 1). The amylose content in the starch of the WT was 15.13%.
The starch from L151, L224 and L259 contained 17.42, 18.73 and 20.44% amylose, respectively.
2.6. Starch granule size and morphology
To quantify the alteration in starch granule size, a granule size distribution assay was performed (Fig. 6-A). The
starch granule size of the transgenic plants displayed different patterns with respect to the WT. Two peaks
appeared at approximately 14 and 80 μm in the three transgenic lines, whereas the WT displayed a unimodal
pattern with a single peak at approximately 14 μm. The diameters of the starch granules from the WT were less
than 50 μm. In contrast, the transgenic lines exhibited broader granule size distributions than the WT, i.e., they
contained a new fraction of starch granules with larger sizes of 50-200 μm. As shown in Fig. 6-B, the mean
volume diameters (MVD) of the WT was 17.02 μm, and the MVD of the transgenic plants surpassed that of WT
due to the existence of the new fraction of starch granules.
The starch granule morphology was examined by scanning electron microscopy (SEM) (Fig. 6- insets). The
transgenic plants contained some extra larger starch granules compared with the WT, which was consistent with
the results of their granule size distributions. No other significant morphological alterations were observed in the
transgenic lines. Each sample contained multiple granular shapes but the characteristic oval shape was
maintained and no fissures in the granules were observed. These morphological characteristics are similar to
those observed by Noda et al. (1995) for sweetpotato. Some incompletely-spherical and polygonal granules
were detected in each sample, which were probably caused by immature development or physical damage during
granule purification.
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2.7. Chain length distribution
To further understand the role of IbAATP in starch biosynthesis, starch from the transgenic plants and WT were
debranched and the glucan chains were quantitatively analyzed using high-performance anion-exchange
chromatography with pulsed amperometric detection (HPAEC-PAD). Amylopectin chains with a degree of
polymerization (DP) of 5-70 were calculated for the transgenic and WT plants based on their peak areas (Fig. 7-A).
The CLD of each line shared a similar pattern with the WT. A trough appeared at DP 8, and two peaks were
evident at approximately DP 14 and DP 47. This pattern is consistent with previous findings (Zhou et al. 2015).
The value for the amylopectin glucan chain of the WT was subtracted from the corresponding value for the
transgenic lines to generate the CLD difference models (Fig. 7-B). In the transgenic lines, chains with DP values
of 5-9 and 24-40 were decreased, whereas the DP 10-23 chains were increased. This result indicated that
IbAATP plays a role in amylopectin biosynthesis, especially in α-1,4 glucan chain elongation which is under the
concerted control of soluble starch synthase (SS), starch branching enzyme (SBE) and starch debranching enzyme
(DBE).
2.8. Starch pasting property
A rapid viscosity analyzer (RVA-Newport Super 3) was used to examine whether the overexpression of IbAATP
has an impact on the starch viscosity properties. The results showed that starch from the transgenic lines had a
lower peak viscosity (PV), hot paste viscosity (HV) and final viscosity (FV) than did the WT (Table 2). In
contrast, the setback (SB) value and pasting temperature (PT) were higher in the transgenic lines than in the WT.
2.9. Expression of starch biosynthetic genes and enzyme activity assay
To understand how IbAATP regulates starch synthesis in sweetpotato, the expressed mRNA levels of 13 other
starch biosynthetic genes (IbPGM, IbAGP-sTL1, IbAGP-sTL2, IbAGP-TLI, IbGBSSI, IbSSI, IbSSII, IbSSIII,
IbSSIV, IbSBEI, IbSBEII, IbIsa1 and IbPUL) in the three transgenic lines and WT were investigated using
qRT-PCR (Fig. 8). These 13 genes were divided into three groups according to their individual functions in
starch biosynthesis. Group I members (IbPGM, IbAGP-sTL1, IbAGP-sTL2 and IbAGP-TLI) may exert control
over the total carbon flux into the starch biosynthesis pathway and thus influence the starch content. Group II
(IbGBSSI) contributed to amylose synthesis, and group III members (IbSSI, IbSSII, IbSSIII, IbSSIV, IbSBEI,
IbSBEII, IbIsa1 and IbPUL) were responsible for the synthesis of amylopectin (Nakamura 2002; Tetlow et al.
2004; Streb et al. 2009; Tetlow 2010). Every gene in groups I and II was upregulated. In group III, IbSSI to IV
played a key role in amylopectin chain elongation, and IbSSI, IbSSII and IbSSIV were upregulated whereas IbSSIII
was slightly down-regulated. The transcripts of the genes encoding the starch branching enzyme (IbSBEI,
IbSBEII) and the debranching enzyme (IbIsa1, IbPUL) were enhanced in this group. The activities of the major
enzymes (SS, AGPase, GBSS, SBE) involved in the starch biosynthesis were assayed in the storage roots of the
transgenic plants and WT. All four enzymes exhibited higher activities in the transgenic plants than in the WT.
This result was consistent with the altered transcript levels of the genes that encode these four enzymes.
3. Discussion
In heterotrophic organs, the carbon precursors and ATP needed for anabolic processes are mainly imported from
the cytoplasm (Emes and Neuhaus 1997; Winkler and Neuhaus 1999). The levels of ATP or ADP-Glc are key
targets for genetic manipulation when attempting to accelerate starch biosynthesis in amyloplasts. ATP uptake
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by amyloplasts is mediated by a plastidic ATP/ADP transporter (AATP) (Schünemann et al. 1993).
In the present study, we isolated the IbAATP from the high-starch sweetpotato line Xu 781, and sequence
analysis indicated that IbAATP is a previously unreported member of the plastidic ATP/ADP transporter gene
family. A high transcript level of IbAATP was observed in the storage roots (heterotrophic) and mature leaves
(autotrophic). This result, as explained by Tjaden et al. (1998), first indicates the high demand for the ATP
supply to support starch synthesis in these two organs and second, suggests that the production of ATP via
glycolysis in the amyloplasts of storage roots is not sufficient for the anabolic process in this organelle. The
strong induction of the IbAATP transcript by exogenous sucrose indicates that IbAATP may be tightly associated
with starch biosynthesis in sweetpotato and may serve as a means to reprogram chloroplasts into starch
accumulating ATP-importing storage plastids (Koch 1996; Reiser et al. 2004). Once the sugar level in the
cytosol is higher than actually needed, IbAATP will be strongly induced, which couples this high sugar level by
importing more ATP into the plastids, thus triggering the starch biosynthesis. The results of the spatial
expression pattern of IbAATP and its induction by sucrose demonstrate that one potential way to increase the
efficiency of starch biosynthesis in sweetpotato storage roots may rely on elevating the sugar levels in the cytosol
as well as enhancing the expression of IbAATP.
The overexpression of AtAATP1 in potato leads to an increase in starch accumulation in tubers because more
ATP is imported from the cytosol into the stroma, which facilitates the synthesis of ADP-Glc (Tjaden et al. 1998).
In the current study, the starch content in the transgenic plants was significantly enhanced (Table 1). The
overexpression of IbAATP could also cause the increased ATP import into amyloplasts, energizing the pivotal
AGPase reaction in starch biosynthesis. Thus, the genes encoding the large and small subunits of AGPase in
sweetpotato were strongly induced (Fig. 8) and the enzymatic activity of AGPase was significantly enhanced in
the transgenic plants (Fig. 9). A large amount of the ultimate precursor for starch synthesis, ADP-Glc, would
accumulate. Meanwhile, the consumption of glucose-1-phosphate (Glc1P) in the AGPase reaction required the
accelerated conversion of glucose-6-phosphate (Glc6P) to Glc1P, which was catalyzed by phosphoglucomutase
(PGM), and consequently, the transcription of IbPGM was upregulated (Fig. 8). These results suggest that the
overexpression of IbAATP increased the starch content by enhancing the ATP supply into amyloplasts, which
further resulted in the increased production of precursors (ADP-Glc and Glc1P) for starch biosynthesis.
The overexpression of IbAATP increased the amylose content in transgenic sweetpotato, which is similar to the
result obtained in a previous study on potato (Tjaden et al. 1998). This finding could be explained by the
differential regulation of genes controlling amylose or amylopectin synthesis. In general, granule-bound starch
synthase (GBSS) is responsible for amylose synthesis and soluble starch synthases (SSI to IV) are responsible for
the synthesis of amylopectin (Geigenberger 2011). As shown in Fig. 8, the transcript level of IbGBSSI was
significantly increased in the transgenic lines, with L259 showing the greatest up-regulation, i.e., 5.5-fold higher
than that of the WT. For SSI to IV, three genes (SSI, SSII and SSIV) were upregulated whereas the SSIII showed
slight down-regulation. On the enzymatic level, the increase in the activity of GBSS in the transgenic plants was
greater than that of SS, which facilitated the synthesis of amylose. Meanwhile, it was previously indicated that
granule-bound starch synthase (GBSS) displayed a lower affinity for ADP-Glc than soluble starch synthase (SS)
(Smith et al. 1997; Szydlowski et al. 2009). Tjaden et al. (1998) showed that the reduced amylose content in the
potato StAATP1 antisense lines was due to the uneven limitation on the activity of GBSS and SS when the
concentration of ADP-Glc was reduced. In the current study, the enhanced level of AGPase led to a higher
concentration of ADP-Glc, which would stimulate the activity of GBSS more than the activity of SS due to their
different affinities for ADP-Glc. As a consequence, the amylose content was increased in the transgenic lines.
These results indicate that in the transgenic plants, the GBSS was activated more than the soluble starch synthases,
thus leading to an improved level of amylose.
The IbAATP-overexpressing sweetpotato plants exhibited different granule size distribution patterns and
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increased MVD of starch granules (20.6-31.3% enlargement) compared with the WT (Fig. 6). In potato, the size
distribution of starch granules from AATP sense transgenic plants is not substantially different from that of the WT
(Geigenberger et al. 2001). It was also reported that alterations in the activity of starch biosynthetic enzymes
(such as SBE and GBSS) can also affect the starch granule size (McDonald et al. 1991; Buléon et al. 1997). In
the present study, the MVD of starch granules from transgenic plants was increased mostly due to the occurrence
of a new fraction of larger starch granules (Fig. 6-B). This extra part of starch granules implies that there may be
an alteration in starch granule initiation. A large body of evidence have illustrated that the soluble starch
synthase III, IV and isoamylase are involved in this process (Burton et al. 2002; Bustos et al. 2004; Roldán et al.
2007; Szydlowski et al. 2009). Here, the transcript levels of IbSSIII, IbSSIV and IbIsa1 were differentially
altered in the transgenic lines compared with the WT, thus providing a possible explanation for the existence of an
extra fraction of starch granules. Therefore, the changes in granule size distribution in transgenic plants are the
combined effects of the altered expression levels of starch biosynthetic genes (IbSBE, IbGBSSI, IbSSIII, IbSSIV
and IbIsa1).
The amylopectin composition of the transgenic plants contained more chains with DP 10-23 and less chains
with DP 5-9 and 24-40 (Fig. 7-B). This pattern of CLD alteration was similar to that of the rice SSIIIa mutant
and opposite to that of the rice beIIb mutant (Satoh et al. 2003; Fujita et al. 2007). In the rice SSIIIa mutant, the
CLD was altered due to the deficiency in SSIIIa and the concurrent enhanced SSI activity. Interestingly, the
expression of IbSSI was upregulated, whereas that of IbSSIII was down-regulated in the transgenic plants in this
study, similar to the correlation between SSIII and SSI activity in the rice SSIIIa mutant. Previous studies
indicated that SSI and SSII mainly synthesize amylopectin chains with DP 8-12 (short chains) and DP 13-25
(intermediate chains), respectively (Fujita et al. 2006; Kitahara et al. 2007; Takahata et al. 2010). Thus, the
higher proportion of chains with DP 10-23 in transgenic plants was most likely due to the enhanced expression
levels of IbSSI and IbSSII. Furthermore, the transcript level of IbSBE was significantly increased in the transgenic
plants, which may also contribute to the altered starch structure, as observed in previous studies (Satoh et al. 2003;
Kitahara et al. 2007; Burtardo et al. 2011; Brummell et al. 2015). Therefore, the apparent alteration in the
amylopectin structure in the transgenic sweetpotato was the mutual effects of several starch biosynthetic genes,
such as IbSSI to IV and IbSBE.
RVA analysis showed that the transgenic plants had lower PV, HV, and FV values and higher SB and PT values
than those of the WT. These findings may be due to the starch of these transgenic plants containing more
intermediate chains (DP 13-25) and fewer short chains (DP 5-10). Low values for PV, HV and FV were also
found in wheat with a higher proportion of DP 13-23 chains, a reduced number of DP 8-12 chains, and an
increased amylose content (McMaugh et al. 2014). In high-amylose sweetpotato starch, the PT and SB values
were elevated, whereas the breakdown value was reduced (Zhou et al. 2015). Therefore, the altered pasting
properties of starch in the current study were determined collectively by many factors such as the starch
composition and starch structure.
4. Conclusion
In conclusion, the IbAATP was successfully isolated from sweetpotato. Its overexpression significantly increased
the starch and amylose content and altered the starch structure. These alterations were due to the differential
expression of starch biosynthetic genes caused by the overexpression of IbAATP. These results demonstrate the
explicit role of IbAATP in the starch biosynthesis of sweetpotato, and this gene has the potential to be used for
improving starch content and quality in sweetpotato and other plants.
5. Materials and methods
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5.1. Plant materials
The sweetpotato line Xu 781 (a high-starch line) and cv. Lizixiang (a low-starch cultivar) were cultivated in a
greenhouse under a regimen of 16 h light and 8 h darkness (28°C). Xu 781 was employed for the isolation of the
IbAATP gene and Lizixiang was used to characterize the function of this gene.
5.2. Isolation of IbAATP cDNA and genomic DNA sequences
Total RNA was extracted from the leaves of Xu 781 using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing,
China) and then transcribed into first strand cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit
(TaKaRa Biotechnology (Dalian) Co., Ltd, China). A pair of degenerate primers (DA-F/R, Table S1) was
designed according to the most homologous region resulting from multiple alignments of AATP from different
plant species, and a fragment was amplified from the first strand cDNA. Then, RACE method was conducted to
obtain the full-length cDNA. Gene specific primers (GSPs) were used for the 5’ RACE (5GSP1, 2 and 3) and 3’
RACE (3GSP1 and 2). The genomic sequence of IbAATP was amplified with primers GA-F/R using genomic
DNA extracted from the leaves of Xu 781 as a template. The sequences of the primers used in this study are
listed in Table S1.
5.3. Sequence analysis of IbAATP
The open-reading frame (ORF) of the cloned IbAATP gene was predicted with ORF Finder
(http://www.ncbi.nlm.nih.gov/projects/gorf/). The homology of the IbAATP protein was identified using protein
Blast in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The molecular weight and pI of the IbAATP
protein were calculated using http://web.expasy.org/compute_pi/. The subcellular localization of IbAATP was
predicted using TargetP (version 1.1) and ChloroP (version 1.1) (http://www.cbs.dtu.dk/services/). Multiple
sequence alignment between IbAATP and other AATP proteins was conducted using DNAMAN software. The
genomic structure of IbAATP was analyzed using the Spidey Program (http://www.ncbi.nlm.nih.gov/spidey/).
The phylogenic tree was constructed using MEGA 6.0 software with the neighbor-joining (NJ) method. The
transmembrane helices in the AATP protein were detected using TMHMM Server (v. 2.0,
http://www.cbs.dtu.dk/services/TMHMM/). The accession numbers of the AATP proteins used in the multiple
sequence alignment and phylogenic tree are listed in Table S2.
5.4. Expression of IbAATP in sweetpotato
Total RNA was isolated from five different tissues (storage root, fibrous root, stem, leaf and petiole) of
sweetpotato and first strand cDNA was synthesized using the PrimeScriptTM RT reagent Kit with gDNA Eraser
(Perfect Real Time) (TaKaRa Biotechnology (Dalian) Co., Ltd, China). qRT-PCR was conducted to determine
the transcript levels of IbAATP using SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa Biotechnology (Dalian)
Co., Ltd, China) on a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers
used to amplify IbAATP and IbActin (endogenous control) are listed in Appendix B.
The response of IbAATP to exogenous sucrose was investigated as described by Wang et al. (2001) with some
modifications. Leaf-petioles (10 cm) from Xu 781 were supplied with water or 175 mmol L-1 sucrose in
darkness at 28°C after being cultured in water in the dark for 1 day. qRT-PCR was conducted to determine the
transcript levels of IbAATP in the cuttings harvested at different time points (0, 2, 4, 6, 12, 24 and 48 h) after
treatment.
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The response of IbAATP to the circadian rhythm was examined as described by Leterrier et al. (2008) with
slight modifications. Six in vitro-grown plants of Xu 781 were acclimated to a 16-h-light/8-h-dark regimen in a
growth chamber at 28°C for 1 month prior to modifying the light conditions. RNA was extracted from these
plants that were harvested at different time points: 8 h (P1) and 16 h in the light (0 h in the dark, P2), and 4 h (P3),
8 h (P4), 16 h (P5) and 24 h (P6) in the dark. Subsequently, the samples were subjected to qRT-PCR to
determine the expression levels of IbAATP. The IbGBSSI gene, the expression of which has been reported to
follow a circadian rhythm (Wang et al. 2001; Wang et al. 2004), served as a positive control.
5.5. Subcellular localization
To construct the IbAATP-GFP expression vector, the ORF of IbAATP was amplified from first strand cDNA using
primers 83A-F/R. Then, the sequence-validated ORF fragment was ligated into the pMDC83 vector. This
recombinant vector and the native pMDC83 vector were then introduced into the Agrobacterium tumefaciens
strain EHA105 using the freeze-thaw method, and the positive bacterial strains were used for transient expression
in Nicotiana benthamiana leaf epidermal cells according to the method of Strasser et al. (2007). In addition,
onion epidermal cells were used for the subcellular localization of IbAATP. The coating of the recombinant
IbAATP-GFP vector with gold particles and the bombardment of onion epidermal peels with the coated vector
was conducted as described by Singh et al. (2012). After co-cultivation, the agroinfiltrated tobacco leaves and
the bombarded onion epidermal peels were visualized using a laser scanning confocal microscope (Nikon Inc.,
Melville, NY).
5.6. Production of transgenic sweetpotato plants
The ORF of IbAATP was amplified using primers OA-F/R and then inserted into pBI121 between BamHI and
SacI to replace the glucuronidase (gusA) gene. Subsequently, the expression cassette 35S-IbAATP-NOS was
excised from the pBI121-IbAATP vector using EcoRI and PstI and then ligated between the same cleavage sites in
pCAMBIA3301 to generate the recombinant overexpression vector. This plasmid was transfected into the A.
tumefaciens strain EHA105. Plant transformation and regeneration were performed as described by Liu et al.
(2013) using embryogenic suspension cultures of Lizixiang established as described by Liu et al. (2001). The
putatively transgenic sweetpotato plants were identified by histochemical GUS assay according to Jefferson et al.
(1987). Genomic DNA was extracted from the leaves of the GUS-positive plants and PCR amplifications were
performed with primers T35-F and TA-R listed in Appendix B.
The transgenic and WT plants were transplanted in 19-cm diameter pots containing a mixture of soil,
vermiculite and humus (1:1:1, v/v/v) in a greenhouse. The 25-cm long cuttings were further transplanted into a
field at the end of June. Each plant line (transgenic or WT) consisted of 20 plants with a 25-cm spacing within
rows and an 80-cm spacing between rows. All the experiments were conducted under normal field conditions.
Storage roots were harvested in mid-October and used for starch content and quality analyses.
5.7. Quantification of starch content and composition
The starch content in the storage roots of the transgenic and WT plants was analyzed according to the method of
Smith and Zeeman (2006). Starch was isolated from sweetpotato storage roots as described by Zhao et al. (2011).
Fresh slices of sweetpotato tuberous roots were suspended in distilled water and crushed in a blender and the
slurry was filtered through a 100-μm sieve. The supernatant was discarded after sedimentation of the starch
granules. This process was repeated three times, and the starch samples were dried in a convection oven at 40°C
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for 2 days. The amylase/amylopectin ratio in the starch was then quantified using an amylose/amylopectin assay
kit (Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, Ireland) according to the
manufacturer’s instructions (http://www.megazyme.com).
5.8. Analysis of starch granule size and morphology
The analysis of granule size distribution was conducted as described by Zhou et al. (2015) using starch granules
isolated from the storage roots of the transgenic plants and the WT. The Master-size 2000 laser diffraction
instrument (Malvern Instruments Ltd., Worcestershire, UK) was used in the wet-well mode. The starch was
added to the reservoir and sonicated for 30 s at 6 W until an obscuration value of 12-17% was achieved. The
refractive indices used for water and starch were 1.330 and 1.50, respectively. The results of the particle size
distribution are presented as diameter versus volume (Blazek and Copeland 2008). To examine whether the
starch granule morphology was altered in different transgenic lines, the starch granules were gold coated after they
were spread on a metal stub and then observed under a scanning electron microscope.
5.9. Analysis of the chain length distribution
The chain length distribution (CLD) measurement was performed according to Zhou et al. (2015). Starch from
the transgenic plants and WT was digested using Pseudomonas amyloderamosa isoamylase (I5284;
Sigma-Aldrich Shanghai Trading Co. Ltd., China) and the CLD of amylopectin was analyzed using
high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD;
Dionex-ICS 3000; Dionex Corporation, Sunnyvale, CA, USA).
5.10. Measurement of starch pasting properties
The pasting properties of the starch were analyzed using a rapid viscosity analyzer (model RVA-Super 3; Newport
Scientific Pty. Ltd., Warriewood, Australia). The starch was suspended in distilled water (10% w/v, dry weight
basis, 25 mL) and tested using the integrated sweetpotato program. The temperature of the starch slurry was
raised from 30 to 95°C at a rate of 5°C min-1 and held at 95°C for 6 min, then lowered to 50°C at the same rate
and held for 10 min. The rotating speed of the paddle remained constant (160 rpm) throughout the analysis,
excluding the speed of 960 rpm applied during the first 10 s.
5.11. Expression analysis of starch biosynthetic genes
The transcript levels of IbAATP and 13 key genes in the starch biosynthetic pathway in the storage root of
transgenic plants and WT were investigated using qRT-PCR. The 13 genes included IbPGM, IbAGP-sTL1 and 2
(which encodes the two small subunits of IbAGPase), IbAGP-TLI (which encodes a large subunit of IbAGPase),
IbGBSSI, IbSSI, IbSSII, IbSSIII, IbSSIV, IbSBEI, IbSBEII, IbIsa1 and IbPUL. The primers used to amplify these
genes are listed in Appendix B.
5.12. Enzyme activity assay in storage roots
The activity of the four major starch biosynthetic enzymes (SS, AGPase, GBSS and SBE) in the storage roots of
the transgenic plants and WT were measured as described by Nakamura et al. (1989) with some modifications.
The homogenate of the storage root samples were centrifuged at 10000×g for 5 min. The resulting supernatant
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was used for the activity assay of SS, AGPase and SBE, and the precipitate was resuspended for the measurement
of GBSS activity. One unit of activity (SS, GBSS and AGPase) was defined as the formation of 1 nmol ADP per
min at 30oC and 1 unit of SBE activity was defined as the amount of enzyme required to increase the
spectrophotometric absorbance by 1 unit in 1 min.
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Table 1 Quantification of starch content and composition
Sample
Starch content (mg g-1 FW)
Amylose content (%)
WT
121.17+1.76
15.13+0.11
L151
190.58+1.94**
17.42+0.19**
L224
153.57+2.67**
18.73+0.21**
L259
165.85+1.72**
20.44+0.23**
Data are presented as the mean+SE (n=3).
**
indicates a significant difference versus the WT at P<0.01, based on Student’s t-test.
The same as below.
Table 2 RVA analysis of starch
Hot paste
Sample
Peak viscosity (cP)
Final
Breakdown (cP)
viscosity (cP)
WT
*
3533.00 + 10.02
Peak time
Pasting
Setback (cP)
Viscosity (cP)
(min)
o
temperature ( C)
2212.00 + 29.19
1321.00 + 19.35
2992.67 + 30.90
780.67 + 1.86
10.3996
66.80 + 0.17
L151
3414.67 + 4.84**
2010.67 + 39.20*
1404.00 + 38.37**
2976.67 + 12.47
966.00 + 51.48*
10.5330
67.43 + 0.22
L224
3288.00 + 21.08**
2020.33 + 17.33*
1267.67 + 26.97
2978.33 + 39.62
958.00 + 41.05*
10.9330
71.10 + 0.13**
L259
3330.33 + 11.98**
2131.67 + 44.38
1198.67 + 32.41*
2957.67 + 12.41
826.00 + 41.67
10.3330
68.93 + 0.20**
indicate a significant difference versus the WT at P<0.05, based on Student’s t-test.
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Fig. 1 Multiple sequence alignment of plastidic ATP/ADP transporters from Ipomoea batatas (IbAATP), Solanum lycopersicum
(SlAATP), Solanum tuberosum (StAATP1), Arabidopsis thaliana (AtAATP1, 2), Zea mays (ZmAATP), Oryza sativa (OsAATP1, 2)
and Rickettsia prowazekii (TLCRp).
by gray background.
Identical residues are denoted by dark blue shading, and conserved amino acids are indicated
The ten transmembrane helices of IbAATP predicted by TMHMM are outlined. Five highly conserved
motifs (FLKT, AELWG, FANQIT, AYG(I/V)S(I/V)NLVE, and (L/I)GKSGGA(L/I)IQ) present in the plant and bacterial ATP/ADP
transporter proteins are indicated in red boxes.
cleavage site is denoted in the blue box.
with a black arrow.
The conserved N-terminal motif in dicotyledonous AATPs harboring the TP
The cleavage site of the IbAATP transit peptide predicted by ChloroP (ver. 1.1) is marked
The accession numbers of these proteins are listed in Appendix C.
Fig. 2 Phylogenetic analysis of plastidic ATP/ADP transporters from Ipomoea batatas (IbAATP) and other plant species. Sequences
were from Solanum lycopersicum (SlAATP), Solanum tuberosum (StAATP1), Vitis vinifera (VvAATP), Mesembryanthemum
crystallinum (McAATP1), Cucumis sativus (CsAATP), Medicago truncatula (MtAATP), Manihot esculenta (MeAATP1, 2), Ricinus
communis (RcAATP), Arabidopsis thaliana (AtAATP1, 2), Oryza sativa (OsAATP1, 2) and Zea mays (ZmAATP).
numbers of these proteins are listed in Appendix C.
The accession
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Expression analysis of IbAATP in sweetpotato.
using qRT-PCR.
L, leaf; S, stem; P, petiole; SR, storage root; FR, fibrous root. B, Effect of exogenous sucrose treatment on IbAATP
transcript accumulation in leaf-petioles.
dark conditions.
A, Transcript level of IbAATP in different tissues of sweetpotato detected
C, Top: time settings used to examine the circadian rhythm during alternating light and
The open bars represent the 16-h light period, the solid bars correspond to the 8-h dark period, and the hatched
bars indicate the extended dark period that would otherwise be the light period.
different time points under varying light conditions.
which were set to 1.0 in A, B and C.
Bottom: transcript levels of IbGBSSI and IbAATP at
The results are expressed as relative values with respect to “L”, “0 h” and “P1”,
Data are presented as means+SD (n=3).
control at P<0.05 and <0.01, respectively, based on Student’s t-test.
Fig. 4 Subcellular localization of the IbAATP protein.
*
and
**
indicate a significant difference versus the
The same as below.
Upper row: confocal images of green fluorescence of the IbAATP-GFP
fusion protein are shown in Nicotiana benthamiana leaf hypodermal cells, which co-localized with the autofluorescence of
chloroplasts.
Lower row: IbAATP-GFP is observed as small spots and clava in the onion peel epidermal cells. Scale bars=50 μm.
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Fig. 5 Transcript level of IbAATP in the tuberous roots of the transgenic plants and WT.
with respect to the WT, which was set to 1.0.
Data are presented as means+SD (n=3).
The results are expressed as relative values
**
indicates a significant difference versus
the WT at P<0.01, based on Student’s t-test.
Fig. 6
Size distribution and morphology of starch granules. A, size distributions and scanning electron micrographs (insets) of
starch granules from the transgenic plants and WT.
The dotted line in the insets indicates a length of 50.0 µm.
B, mean volume
diameter (MVD) of starch granules from the transgenic plants and WT are divided into two parts: MVD of starch granules with
diameters >50 μm and <50 μm.
Fig. 7
Amylopectin chain length distribution of the transgenic plants and the WT.
A, chain length distributions (CLDs) of the
starch isolated from the transgenic lines and the WT after normalization to the total peak area.
B, differences in the CLD between
the transgenic lines and the WT were calculated as follows: the normalized CLD value for each transgenic line minus the value
obtained for the WT.
Journal of Integrative Agriculture
Advanced Online Publication 2015
Fig. 8
Doi: 10.1016/S2095-3119(15)61192-3
Transcript levels of starch biosynthetic genes in the tuberous roots of the transgenic plants and WT.
expressed as relative values with respect to the WT, which was set to 1.0.
Fig. 9
Enzyme activities of SS, AGPase, GBSS, SBE in the storage roots of the transgenic plants and WT.
The results are