Starch Synthesis

JBA-06696; No of Pages 20
Biotechnology Advances xxx (2013) xxxxxx
Contents lists available at SciVerse ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Starch biosynthesis, its regulation and biotechnological approaches to improve

crop yields
Abdellatif Bahaji a,1, Jun Li a,1, ngela Mara Snchez-Lpez a, Edurne Baroja-Fernndez a,
Francisco Jos Muoz a, Miroslav Ovecka a,b, Goizeder Almagro a, Manuel Montero a, Ignacio Ezquer a,
Ed Etxeberria c, Javier Pozueta-Romero a,
a
b
c
Instituto de Agrobiotecnologa (CSIC/UPNA/Gobierno de Navarra), Mutiloako etorbidea z/g, 31192 Mutiloabeti, Nafarroa, Spain
Centre of the Region Han for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacky University, lechtitel 11, CZ-783 71 Olomouc, Czech Republic
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA
a r t i c l e
i n f o
Available online xxxx

Keywords:
ADPglucose
CalvinBenson cycle
Carbohydrate metabolism
Genetic engineering
Microbial volatiles
MIVOISAP
Plantmicrobe interaction
Endocytosis
Starch futile cycling
Sucrose synthase
a b s t r a c t
Structurally composed of the glucose homopolymers amylose and amylopectin, starch is the main storage carbohydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells.
Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both a
cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including
production of adhesives, biodegradable materials, and rst-generation bioethanol. This review provides an update on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophic
organs, and provides emerging information about the networks regulating them and their interactions with the
environment. Special emphasis is given to recent ndings showing that volatile compounds emitted by microorganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starch
metabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species.
Finally we present some potential biotechnological strategies for enhancing starch content.
2013 Elsevier Inc. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
Proposed starch biosynthetic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
2.1.
Starch biosynthesis in leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
2.1.1.
A classic model of starch biosynthesis according to which the starch biosynthetic pathway is linked to the CalvinBenson cycle by means of
plastidial phosphoglucose isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
2.1.2.
Models of starch biosynthesis according to which the starch biosynthetic pathway is not linked to the CalvinBenson cycle by
means of plastidial phosphoglucose isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
2.2.
Starch biosynthesis in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
2.2.1.
Mechanisms of sucrose unloading in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
2.2.2.
Proposed mechanisms of sucrosestarch conversion in heterotrophic cells . . . . . . . . . . . . . . . . . . . . . . . . . . .
0
Abbreviations: ADPG, ADPglucose; AGP, ADPG pyrophosphorylase; AGPP, ADPG pyrophosphatase; A/N-inv, alkaline/neutral invertase; BT1, Brittle 1; F6P, fructose-6-phosphate; G1P,
glucose-1-phosphate; G6P, glucose-6-phosphate; GBSS, granule bound starch synthase; GPT, glucose-6-phosphate translocator; GWD, glucan, water dikinase; HvBT1, Hordeum vulgare
BT1; MCF, mitochondrial carrier family; MVs, microbial volatiles; NPP, nucleotide pyrophosphatase/phosphodiesterase; MIVOISAP, MIcrobial VOlatiles Induced Starch Accumulation Process; NTRC, NADP-thioredoxin reductase C; OPPP, oxidative pentose phosphate pathway; Pi, orthophosphate; 3PGA, 3-phosphoglycerate; pHK, plastidial hexokinase; pPGI, plastidial
phosphoglucoseisomerase; pPGM, plastidial phosphoglucomutase; PPi, inorganic pyrophosphate; pSP, plastidial starch phosphorylase; RSR1, Rice Starch Regulator1; SuSy, sucrose
synthase; SS, starch synthase; T6P, trehalose-6-phosphate; Trx, thioredoxin; UDPG, UDPglucose; UGP, UDPG pyrophosphorylase; WT, wild type; ZmBT1, Zea mays BT1.
Corresponding author. Tel.: +34 948168009; fax: +34 948232191.
E-mail address: javier.pozueta@unavarra.es (J. Pozueta-Romero).
1
These authors contributed equally to this work.
0734-9750/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.06.006
Please cite this article as: Bahaji A, et al, Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields, Biotechnol
Adv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.06.006
A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx
3.
4.
Microbial volatiles promote both growth and accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants
Biotechnological approaches for increasing starch content in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Enhancement of AGP activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Increasing supply of starch precursors to the amyloplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Increasing supply of sucrose to heterotrophic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Enhancement of sucrose breakdown within the heterotrophic cell . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
Enhancement of expression of starch synthase class IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.
Blocking starch breakdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.
Altering the expression of global regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8.
Enhancing trehalose-6-phosphate content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.9.
Potential strategies for increasing starch content in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . .
4.9.1.
Over-expression of BT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.9.2.
Blocking the activity of ADPG breakdown enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.9.3.
Ectopic expression of SuSy in the plastid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.9.4.
Blocking the synthesis of storage proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Conclusions and main challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Starch is the main storage carbohydrate in vascular plants. Its abundance as a naturally occurring compound of living terrestrial biomass is
surpassed only by cellulose, and represents the primary source of calories in the human diet. Because starch is the principal constituent of
the harvestable organ of many agronomic plants, its synthesis and accumulation also inuences crop yields.
Synthesized in the plastids of both photosynthetic and nonphotosynthetic cells, starch is an insoluble polyglucan produced by
starch synthase (SS) using ADP-glucose (ADPG) as the sugar donor molecule. Starch has two major components, amylopectin and amylose, both
of which are polymers of -D-glucose units. Amylose is a linear polymer
of up to several thousand glucose residues, whereas amylopectin is a
larger polymer regularly branched with -1,6-branch points. These
two molecules are assembled together to form a semi-crystalline starch
granule. The exact proportions of these molecules and the size and
shape of the granule vary between species and between organs of the
same plant. The diversity of both composition and physical parameters
of starches from different botanical sources gives rise to their diverse
processing properties and applications in both non-food sectors such as
sizing agents in textile and paper industry, adhesive gums, and biodegradable materials, and in food industries, particularly in bakery, thickening, confectionary and emulsication (Burrell, 2003; Mooney, 2009;
Slattery et al., 2000). Starch is also used as a feedstock for rst generation
bioethanol production (Goldemberg, 2007). From an industrial perspective, the utilization of starch as a cheap and renewable polymer and clean
energy source is becoming increasingly attractive as a consequence of social concerns about industrial wastes generated from fossil fuels.
In general, the harvested parts of our staple crop plants are heterotrophic starch-storing organs. Worldwide annual starch production of
cereal seeds (rice, maize, wheat, barley, etc.) is approximately
2 billion tons, whereas worldwide annual starch production of roots
(e.g. cassava and taro) and tubers (e.g. potato and sweet potato) exceeds 700 million tons (Food and Agriculture Organization of the United Nations, http://faostat.fao.org), much of which is destined to nonfood purposes. In 2012 for instance, 75 million tons of starch were
extracted and used in many industrial applications (http://www.
starch.dk/). The increasing demand of starch from non-food industries,
the continuing rapid increase in the world's population, predicted to
reach 9 billion by 2050, and the growing loss of arable land to urbanization have spurred on efforts to synthesize plants with higher starch
content. Therefore, a thorough understanding of the mechanisms involved in starch metabolism and regulation is critically important for
the rational design of experimental traits aimed at improving yields in
agriculture, and producing more and better polymers that t both
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industrial needs and social demands. However, while much is known

about starch metabolism, there are still major gaps in our knowledge
that prevent breeders and biotechnologists from advancing in their attempts to increase starch content in a predictable way (Chen et al.,
2012).
In this paper, we rst review recent advances in the biochemistry of
starch biosynthesis and its regulation, and provide an update on different
proposed pathways of starch biosynthesis occurring in both autotrophic
and heterotrophic organs. Special emphasis is given to recent ndings
showing that volatile compounds emitted by microorganisms promote
both growth and the accumulation of exceptionally high levels of starch.
Finally, we assess the progress in molecular strategies for increasing
starch in annual crop species by genetic engineering approaches.
2. Proposed starch biosynthetic pathways
Starch is found in the plastids of photosynthetic and nonphotosynthetic tissues. Mature chloroplasts occurring in photosynthetically active cells possess the capacity of providing energy (ATP) and
xed carbon for the synthesis of starch during illumination. By contrast,
production of long-term storage of starch taking place in amyloplasts of
reserve organs such as tubers, roots and seed endosperms depends
upon the incoming supply of carbon precursors and energy from the
cytosol. This difference between the metabolic capacities of chloroplasts
and amyloplasts has led to the generally accepted view that the pathway(s) involved in starch production are different in photosynthetic
and non-photosynthetic cells.
2.1. Starch biosynthesis in leaves
In leaves, a portion of the photosynthetically xed carbon is retained
within the chloroplasts during the day to synthesize starch, which is
then remobilized during the subsequent night to support nonphotosynthetic metabolism and growth by continued export of carbon
to the rest of the plant. Due to the diurnal rise and fall cycle of its levels,
foliar starch is termed transitory starch.
Transitory starch is a major determinant of plant growth (Sulpice
et al., 2009), and its metabolism is regulated to avoid a shortfall of carbon at the end of the dark period. It is typically degraded in a near-linear
manner during darkness, with only a small amount remaining at the
end of the night. When changes in the day length occur that leads to a
temporary period of carbon starvation, the carbon budget is rebalanced
by increasing the rate of starch synthesis, decreasing the rate of starch
breakdown and, consequently, decreasing the rate of growth during
darkness. The importance of starch turnover in plant growth and
biomass production is demonstrated by studies of mutants that are defective in starch synthesis and mobilization. These mutants grow at the
same rate as wild-type (WT) plants under continuous light or during
very long days, but show a large inhibition of growth in short days
(Caspar et al., 1985; Gibon et al., 2004; Lin et al., 1988), which is due
to the transient depletion of carbon during the night.
Leaf starch mainly accumulates in the photosynthetic palisade and
spongy mesophyll cells, although it can also be synthesized in epidermal cells, stomatal guard cells and bundle sheath cells surrounding
the vasculature (Tsai et al., 2009). Various mechanisms have been proposed to describe starch synthesis and degradation in leaves. Given that
starch breakdown has been thoroughly discussed in recent reviews
(Streb and Zeeman, 2012), in the following lines we will focus on describing, analyzing and comparing the proposed mechanisms of starch
synthesis in leaves.
2.1.1. A classic model of starch biosynthesis according to which the starch
biosynthetic pathway is linked to the CalvinBenson cycle by means of
plastidial phosphoglucose isomerase
It is widely assumed that the whole starch biosynthetic process resides exclusively in the chloroplast and segregated from the sucrose biosynthetic process that takes place in the cytosol (Neuhaus et al., 2005;
Stitt and Zeeman, 2012; Streb et al., 2009). According to this classical
view of starch biosynthesis (which is schematically illustrated in
Fig. 1), starch is considered the end-product of a metabolic pathway
that is linked to the CalvinBenson cycle by means of the plastidial
phosphoglucose isomerase (pPGI). This enzyme catalyzes the conversion of fructose-6-phosphate (F6P) from the CalvinBenson cycle into
glucose-6-phosphate (G6P), which is then converted into glucose-1phosphate (G1P) by the plastidial phosphoglucomutase (pPGM).
ADPG pyrophosphorylase (AGP) then converts G1P and ATP into inorganic pyrophosphate (PPi) and ADPG necessary for starch biosynthesis.
These three enzymatic steps are reversible, but the last step is rendered
irreversible upon hydrolytic breakdown of PPi by plastidial alkaline

pyrophosphatase.
pPGI is strongly inhibited by light (Heuer et al., 1982) and by
CalvinBenson cycle intermediates such as erythrose-4-P and 3phosphoglycerate (3PGA) (the latter being an indicator of photosynthetic carbon assimilation) that accumulate during the day at stromal concentrations higher than the Ki values for pPGI (Backhausen
et al., 1997; Dietz, 1985; Kelly and Latzko, 1980; Zhou and Cheng,
2008). Although both these characteristics of pPGI, and the low stromal G6P/F6P ratio occurring during illumination (Dietz, 1985) would
indicate that this enzyme is inactive during illumination (and thus
not involved in transitory starch biosynthesis), genetic evidence
showing that transitory starch biosynthesis occurs solely by the
pPGI pathway has been obtained from characterization of starchdecient pPGI mutants from various species (Jones et al., 1986;
Kruckeberg et al., 1989; Kunz et al., 2010; Niewiadomski et al.,
2005; Yu et al., 2000).
The classic view of leaf starch biosynthesis illustrated in Fig. 1 also
implies that AGP is the sole source of ADPG, and functions as the
major regulatory step in the starch biosynthetic process (Neuhaus
et al., 2005; Stitt and Zeeman, 2012; Streb and Zeeman, 2012; Streb
et al., 2009). This enzyme is a heterotetramer comprising two types of
homologous but distinct subunits, the small (APS) and the large (APL)
subunits (Crevilln et al., 2003, 2005). In Arabidopsis, six genes encode
proteins with homology to AGP. Two of these genes code for small subunits (APS1 and APS2, the latter being in a process of pseudogenization)
and four (APL1APL4) encode large subunits. Whereas APS1, APL1 and
APL2 are catalytically active, APL3 and APL4 have lost their catalytic
properties during evolution (Ventriglia et al., 2008). In Arabidopsis, the
large subunits are highly unstable in the absence of small subunits
(Wang et al., 1998). Therefore, APS1 T-DNA null mutants contain neither
the large nor the small subunit proteins, which result in a total lack of
AGP activity (Bahaji et al., 2011a; Ventriglia et al., 2008).
CO2
Triose-P
Calvin-Benson
cycle
Triose-P
FBP
2
FBP
3
F6P
4
F6P
4
G6P
G6P
5
G1P
Starch
10
G1P
6
ADPG
UDPG
Chloroplast
Sucrose
8
7
Sucrose-P
Cytosol
Fig. 1. Classic interpretation of the mechanisms of starch and sucrose syntheses in leaves. The enzymes are numbered as follows: 1, 1, fructose-1,6-bisphosphate aldolase; 2, 2, fructose
1,6-bisphosphatase; 3, PPi: fructose-6-P 1-phosphotransferase; 4, 4, PGI; 5, 5, PGM; 6, UGP; 7, sucrose-phosphate-synthase; 8, sucrose phosphate phosphatase; 9, AGP; and 10, SS.
According to this view, the starch biosynthetic process resides exclusively in the chloroplast and is segregated from the sucrose biosynthetic pathway that takes place in the cytosol.
AGP is allosterically activated by 3PGA, and inhibited by Pi

(Kleczkowski, 1999). This property makes the production of ADPG highly sensitive to changes in photoassimilate (e.g. 3PGA) availability in the
chloroplast, and helps to coordinate photosynthetic CO2 xation with
starch synthesis in leaves. AGP activity is also subjected to APS redox
regulation (Hendriks et al., 2003; Kolbe et al., 2005; Li et al., 2012;
Michalska et al., 2009). Under oxidizing conditions the two APS subunits
are covalently linked via an intermolecular disulde bridge, thus
forming a stable dimer within the heterotetrameric AGP enzyme (Fu
et al., 1998; Hendriks et al., 2003). Conversely, under reductive conditions APS monomerization is accompanied by activation of AGP activity.
APS dimerization markedly decreases the activity of AGP and alters its
kinetic properties, making it less sensitive to activation by 3PGA and increasing its Km for ATP (Hendriks et al., 2003). In turn, APS
monomerization activates AGP, which is then sensitive to ne control
through allosteric activation by 3PGA.
By examining the correlation between APS redox status, AGP activity
and starch levels in leaves, different works have provided evidence that
transitory starch accumulation is nely regulated by APS posttranslational redox modication in response to environmental inputs
such as light, sugars and biotic stress (Gibon et al., 2004; Hendriks
et al., 2003; Kolbe et al., 2005; J. Li et al., 2011). However, using aps1
Arabidopsis plants ectopically expressing a redox-insensitive, mutated
APS1 form, Li et al. (2012) and Hdrich et al. (2012) have independently
shown that the rates of transitory starch accumulation in these plants
are comparable to that of WT leaves. In addition, Li et al. (2012) reported WT rates of starch accumulation in aps1 plants expressing in the
chloroplast a redox-insensitive AGP from Escherichia coli, and concluded
that post-translational redox modication of APS1 in response to light is
not a major determinant of ne regulation of transitory starch accumulation in Arabidopsis leaves.
Plastidial NADP-thioredoxin reductase C (NTRC) plays an important role in protecting plants against oxidative stress (Pulido
et al., 2010; Serrato et al., 2004). Serrato et al. (2004) and Michalska
et al. (2009) reported that Arabidopsis ntrc mutants grown at
180 mol photons s 1 m 2 are small and their leaves accumulate
low levels of starch when compared with WT leaves. Furthermore,
Michalska et al. (2009) reported that ntrc leaves exhibit a decrease in
the extent of APS1 activation (reduction) by light. These authors
thus concluded that NTRC is a major determinant of both lightdependent APS1 redox status and transitory starch accumulation.
However, Li et al. (2012) have recently found that the size of
Arabidopsis ntrc mutants is similar to that of WT plants when
cultured at 90 mol photons s1 m2. Under these conditions, APS1
redox status and starch accumulation rates in leaves of ntrc mutants
are similar to those of WT leaves, strongly indicating that NTRC plays
a minor role, if any, in the control of both light-dependent APS1 redox
status and transitory starch accumulation in Arabidopsis cultured under
non-stressing conditions.
In vitro assays using heterologously expressed potato and pea AGP
have shown that APS can be activated by plastidial thioredoxins (Trxs) f
and m (Ballicora et al., 2000; Geigenberger et al., 2005), indicating that
these proteins could act as major determinants of APS redox status and
of ne regulation of transitory starch accumulation. However, Li et al.
(2012) found no differences in the rate of starch accumulation between
WT leaves and leaves of homozygous trxf1, trxf2, trxm2 and trxm3 mutants. Furthermore, Thormhlen et al. (2013) reported that starch levels
in trxf1 leaves were only slightly reduced when compared with WT
leaves. In addition, Sanz-Barrio et al. (2013) have shown that APS redox
status in high-starch tobacco leaves overexpressing Trxf and Trxm genes
from the plastid genome is similar to that of WT leaves. The overall
data thus indicate that, individually, plastidial Trxs are not major determinants of APS1 redox status-controlled transitory starch content.
Genetic evidence showing that transitory starch biosynthesis occurs
solely by the AGP pathway has been obtained from the characterization
of leaves with reduced pPGM and AGP activities. For example, leaves of
APS1 and pPGM antisensed potato plants accumulate low levels of starch
when compared with WT leaves (Lytovchenko et al., 2002; MllerRber et al., 1992; Muoz et al., 2005). Furthermore, Arabidopsis pgm
null mutants (Caspar et al., 1985; Koer et al., 2000), adg1-1 mutants
with less than 3% of the WT AGP activity (Lin et al., 1988; Wang
et al., 1998), and APS1 T-DNA null mutants completely lacking AGP
activity (Bahaji et al., 2011a; Ventriglia et al., 2008) accumulate
less than 2% of the WT starch. Further evidence supporting that
AGP plays a crucial role in starch biosynthesis in leaves comes from
alkaline pyrophosphatase-silenced plants of Nicotiana benthamiana,
since their leaves accumulate ca. 60% of the WT starch (George et al.,
2010).
2.1.2. Models of starch biosynthesis according to which the starch
biosynthetic pathway is not linked to the CalvinBenson cycle by means
of plastidial phosphoglucose isomerase
A vast amount of biochemical and genetic data appear to support the
starch biosynthetic pathway illustrated in Fig. 1 according to which (a)
the whole starch biosynthetic process resides exclusively in the chloroplast, (b) pPGI links the CalvinBenson cycle with starch biosynthesis,
and (c) AGP is the sole source of ADPG linked to starch biosynthesis.
However, in recent years an increasing volume of evidence has been
provided that supports the occurrence of additional/alternative starch
biosynthetic pathways involving the cytosolic and plastidial compartments wherein the supply of ADPG is not directly linked to the
CalvinBenson cycle by means of pPGI. Thus, Fettke et al. (2011)
found that the envelope membranes of chloroplasts in mesophyll cells
possess a yet to be identied G1P transport machinery enabling the incorporation of cytosolic G1P into the stroma, which is subsequently
converted into starch by the stepwise AGP and SS reactions. According
to these authors, such mechanism would only allow the accumulation
of 1% of the WT starch, and would explain the occurrence of some
trace amounts of starch in the pPGM mutants (Caspar et al., 1985;
Muoz et al., 2005; Streb et al., 2009).
Kunz et al. (2010) found that leaves of the pgi1-2 null mutant impaired in pPGI activity accumulate 10% of the WT starch content,
which is restricted to bundle sheath cells adjacent to the mesophyll
and stomatal guard cells. This mutant exhibits high G6P transport activity as a consequence of the elevated expression of GPT2 (Kunz et al.,
2010), a gene that codes for a G6P/Pi translocator (GPT) mainly operating in heterotrophic tissues where the imported G6P can be used for the
synthesis of starch and fatty acids or to drive the oxidative pentose
phosphate pathway (Bowsher et al., 2007; Kammerer et al., 1998;
Kang and Rawsthorne, 1996; L. Zhang et al., 2008). According to Kunz
et al. (2010) the unexpected high levels of starch occurring in leaves
of pPGI mutants can be ascribed to high GPT2-mediated incorporation
of G6P into the chloroplast of bundle sheath cells adjacent to the mesophyll and stomatal guard cells, where this hexose-phosphate can be
then converted to starch by means of pPGM, AGP and SS as schematically illustrated in Fig. 2. This interpretation, however, is hardly reconcilable with previous studies carried out by the same group showing
that the starch-decient phenotype of pPGI mutants can be totally
reverted to WT starch phenotype by the ectopic expression of GPT2
(Niewiadomski et al., 2005), and with the fact that leaves of the pgi12/gpt2 double mutant accumulate as much as 60% of the starch occurring in pgi1-2 leaves (Kunz et al., 2010). Furthermore, considering that
(a) most of leaf starch accumulates in the mesophyll cells, (b) microscopic analyses revealed that stomatal guard cells and bundle sheath
cells adjacent to the mesophyll of pPGI mutants accumulate nearly
WT starch content (Kunz et al., 2010; Tsai et al., 2009), and (c) guard
cells of WT leaves possess a G6P transport machinery (Overlach et al.,
1993), it is difcult to understand how guard cells and bundle sheath
cells adjacent to the mesophyll of pPGI mutants can accumulate as
much as 10% of the WT starch as a consequence of high GPT2 activity.
Needless to say, further investigations will be necessary to understand
how and where pgi mutants accumulate ca. 10% of the WT starch.
CO2
Triose-P
CalvinBenson
cycle
Triose-P
1
FBP
3
F6P
4
G6P
5
G6P
GPT
G1P
Starch
10
9
ADPG
Chloroplast
Cytosol
Fig. 2. Suggested model of starch biosynthesis in bundle sheath cells adjacent to the mesophyll and stomatal guard cells of pgi-2 Arabidopsis mutants (from Kunz et al., 2010). Numbering of
enzyme activities is the same as in Fig. 1.
Gerrits et al. (2001) have provided evidence that a sizable pool of

sucrose in the plant cell has a plastidial localization. Consistent with
the occurrence of a plastidial pool of sucrose, Murayama and Handa
(2007) and Vargas et al. (2008) reported the presence of a functional
alkaline/neutral invertase (A/N-inv) inside plastids. Noteworthy,
Vargas et al. (2008) reported that leaves of Arabidopsis mutants impaired
in A/N-inv accumulate ca. 70% of the WT starch content. The same authors hypothesized that chloroplastic sucrose plays a sensing role in a
mechanism of control of carbon partitioning and sucrose/starch ratio
wherein glucose and fructose generated by A/N-inv are sensed through
plastidial hexokinase (pHK) (Giese et al., 2005; Olsson et al., 2003). However, a possibility cannot be ruled out that, as schematically illustrated in
Fig. 3, plastidial sucrose acts as precursor for starch biosynthesis, its
A/N-inv breakdown products being converted into starch by means
of pHK, pPGM and AGP.
Sufcient evidence exists to support the view that a sizable pool of
ADPG in leaves accumulates in the cytosol of photosynthetically competent cells. This hypothesis is compatible with the occurrence of cytosolic
ADPG metabolizing enzymes such as ADPG phosphorylase (McCoy et al.,
2006), glucan synthases (Tacke et al., 1991), sucrose synthase (SuSy) and
ADPG hydrolases (Olejnik and Kraszewska, 2005; Rodrguez-Lpez et al.,
2000), and with the occurrence in the chloroplast envelope membranes
of yet to be molecularly identied ADPG transport machineries whose
activities can account for rates of starch accumulation occurring in leaves
(Pozueta-Romero et al., 1991a). Genetic evidence supporting the occurrence of important ADPG source(s), other than AGP, has been obtained
from different sources. First, leaves of transgenic potato and Arabidopsis
plants ectopically expressing in the cytosol a bacterial ADPG hydrolase
(Moreno-Bruna et al., 2001) accumulate lower ADPG levels than WT
leaves, as determined by HPLC analyses (Bahaji et al., 2011a; BarojaFernndez et al., 2004). Second, ADPG content in the leaves of AGP and
pPGM mutants is comparable to that of WT leaves, as conrmed by
both HPLC (Bahaji et al., 2011a; Muoz et al., 2005) and HPLC:MS
analyses (Baroja-Fernndez et al., 2013). Third, HPLC:MS analyses of

the triple ss3/ss4/aps1 mutant impaired in AGP and SS class III and IV revealed that leaves from this mutant accumulate ca. 40-fold more ADPG
than WT leaves (Ragel, 2012).
SuSy catalyzes the reversible conversion of sucrose and NDP into
fructose and the corresponding NDP-glucose, where N stands for uridine,
adenosine, guanosine, cytidine, thymidine or inosine. Although UDP is
generally considered to be the preferred nucleoside diphosphate for
SuSy, ADP also serves as an effective substrate to produce ADPG
(Baroja-Fernndez et al., 2003, 2012; Cumino et al., 2007; Delmer,
1972; Nakai et al., 1998; Porchia et al., 1999; Silvius and Snyder, 1979;
Zervosen et al., 1998). SuSy is highly regulated at both transcriptional
and post-transcriptional levels (Asano et al., 2002; Ciereszko and
Klezkowski, 2002; Djardin et al., 1999; Fu and Park, 1995; Hardin
et al., 2004; Koch et al., 1992; Pontis et al., 1981; Purcell et al., 1998; for
a review see Kleczkowski et al., 2010). Although this sucrolytic enzyme
is very active in reserve organs, it also expresses in the mesophyll cells
of source leaves (Fu et al., 1995; Wang et al., 1999), its activity in potato
and Arabidopsis leaves greatly exceeding the minimum needed to support normal rate of starch accumulation during illumination (BarojaFernndez et al., 2012; Muoz et al., 2005). Accordingly, a metabolic
model of transitory starch biosynthesis has been proposed wherein (a)
both sucrose and starch metabolic pathways are tightly interconnected
by means of cytosolic ADPG producing enzymes such as SuSy (acting
when cytosolic sucrose transiently accumulates during illumination), and by the action of an ADPG translocator located at the chloroplast envelope membranes, and (b) both AGP and pPGM play an
important role in the scavenging of glucose units derived from starch
breakdown occurring during starch biosynthesis and during the biogenesis of the starch granule (Bahaji et al., 2011a; Baroja-Fernndez
et al., 2005; Muoz et al., 2005, 2006) (Fig. 4). Thus, the net rate of
starch accumulation is determined by the balance between the
rates of SuSy-mediated ADPG synthesis in the cytosol, import of
CO2
CalvinBenson
cycle
Triose-P
Triose-P
1-8
Sucrose
Sucrose
11
Fructose
Glucose
12
F6P
12
4
G6P
5
Starch
10
ADPG
Chloroplast
G1P
Cytosol
Fig. 3. Suggested model of starch biosynthesis where sucrose entering the plastid is broken down by A/N-inv to produce fructose and glucose, the latter being converted to G6P by means of
pHK. G6P is subsequently metabolized to starch by the stepwise reactions of pPGM, AGP and SS. Enzyme activities 110 and 4 and 5 are the same as in legend of Fig. 1. Other enzymes
involved are numbered as follows: 11, A/N-inv; 12, pHK.
cytosolic ADPG to the chloroplast, starch synthesis, starch breakdown and by the efciency with which starch breakdown products
can be recycled back to starch via the coupled reactions of pPGM
and AGP. Accordingly, this view predicts that the recovery toward
starch biosynthesis of the glucose units derived from the starch
breakdown will be decient in pPGM and AGP mutants, resulting in
a parallel decline of starch accumulation.
The occurrence of a substrate or futile cycle as a result of the simultaneous synthesis and breakdown of starch in leaves is not surprising
since pulse-chase and starch-preloading experiments using isolated
chloroplasts (Fox and Geiger, 1984; Stitt and Heldt, 1981), intact leaves
(Scott and Kruger, 1995; Walters et al., 2004), or cultured photosynthetic cells (Lozovaya et al., 1996) have shown that the illuminated
chloroplasts can synthesize and mobilize starch simultaneously. Furthermore, enzymes involved in starch breakdown such as SEX1 and
BAM1 are redox-activated during the light under different environmental conditions (Mikkelsen et al., 2005; Sparla et al., 2006; Valerio et al.,
2010). Futile cycles lead to a net consumption of ATP, which in some instances can be estimated at about 60% of the total ATP produced by the
cell (Alonso et al., 2005; Hill and ap Rees, 1994). Although these cycles
appear to waste energy without any apparent physiological reason,
they form part of the organization of the plant central metabolism,
and contribute to its exibility (Rontein et al., 2002). As presented
above, starch acts as a major integrator in the plant growth that accumulates to cope with temporary starvation imposed by the environment (Sulpice et al., 2009). It is thus conceivable that highly regulated
starch futile cycling may entail advantages such as sensitive regulation
and rapid metabolic channeling toward various pathways in response
to physiological and biochemical needs of the plant. Therefore, due to
the importance of starch in the overall plant metabolism and growth,
it is tempting to speculate that both redundancy of ADPG sources and
starch futile cycling have been selected during plant evolution to
warrant starch production and rapid connection of starch metabolism

with other metabolic pathways.
The postulated starch biosynthetic pathways involving the plastidial
and cytosolic compartments are consistent with still enigmatic results
obtained from experiments carried out more than 50 years ago showing
that 14C in the glucose moiety of sucrose, starch, UDP-glucose and
hexose-Ps is asymmetrically distributed in green leaves exposed to
14
CO2 for a short period of time (Gibbs and Kandler, 1957; Havir and
Gibbs, 1963; Kandler and Gibbs, 1956). Unlike the classic view on transitory starch biosynthesis (Fig. 1) predicting that green leaves exposed to
14
CO2 for a short period of time should synthesize starch with 14C symmetrically distributed in the glucose moiety, the proposed mechanisms
of starch synthesis illustrated in Figs. 24 provide a possible explanation
for solving the above enigma. As shown in Fig. 4, triose-Ps produced in
the CalvinBenson cycle are exported to the cytosol where they can be
channeled into the oxidative pentose-P pathway (OPPP), thereby leading
to a randomization of the carbons giving rise to the asymmetric 14C distribution observed in hexose-Ps, nucleotide-sugars and sucrose upon exposure to 14CO2 for a short period of time. Cytosolic G1P, G6P, sucrose
and/or ADPG will be then transported into the chloroplast and utilized
as precursors for the synthesis of starch molecules possessing glucose
molecules with asymmetric 14C distribution.
Genetic evidence consistent with the occurrence of a link between sucrose and transitory starch metabolism can be obtained from sucrosephosphate-synthase and cytosolic PGM decient plants (Fernie et al.,
2002; Strand et al., 2000), since their leaves contain low levels of both sucrose and starch as compared with WT leaves. Further genetic evidence
can be obtained from sedoheptulose-1,7-bisphosphate overexpressing
plants (Lefebvre et al., 2005; Miyagawa et al., 2001), since their leaves
are characterized by having high levels of both sucrose and starch when
compared with WT leaves. Finally, genetic evidence indicating that leaf
sucrose and starch metabolic pathways are linked by SuSy has been
CO2
CalvinBenson
cycle
Triose-P
Triose-P
OPPP
Sulfolipids
Fatty acids,
OPPP, etc.
Hexose-Ps
1-8
1 - 4
UDPG
GPT
G6P
12
Glucose
15
13
G1P
Sucrose
ADP
16
9
Starch
10
14
ADPG
Chloroplast
Fructose
ADPG
Cytosol
Fig. 4. Suggested interpretation of the mechanism of starch biosynthesis in leaves according to which (a) ADPG is produced in the cytosol and transported to the chloroplast by the action
of a yet to be identied ADPG translocator, (b) AGP and pPGM play an important role in the scavenging of glucose units derived from starch breakdown, and (c) starch metabolism is
connected with other metabolic pathways through the metabolites generated by the starch futile cycle. Enzyme activities 110 and 15 are the same as in legend of Fig. 1. Other enzymes
involved are numbered as follows: 12, pHK; 13, plastidial UGP (Okazaki et al., 2009); 14, plastidial AGPP; 15, pSP (Zeeman et al., 2004); and 16, SuSy. Starch to glucose conversion would
involve the coordinated actions of amylases, isoamylases and disproportionating enzymes (Asatsuma et al., 2005; Fulton et al., 2008; Kaplan and Guy, 2005). Note that starch futile cycling
(indicated with red arrows) may entail advantages such as rapid metabolic channeling toward various pathways (such as fatty acid (Periappuram et al., 2000), OPPP under stress conditions (Zeeman et al., 2004) and sulfolipid biosynthesis (Okazaki et al., 2009)) in response to physiological and biochemical needs. This view predicts that the recovery toward starch biosynthesis of the glucose units derived from the starch breakdown will be decient in pPGM and AGP mutants, resulting in a parallel decline of starch accumulation and enhancement of
soluble sugars content since starch breakdown derived products (especially glucose and G6P) will leak out the chloroplast through very active glucose translocator (Cho et al., 2011) and
GPT2 (which is highly expressed in pgi, pgm and agp mutants, Kunz et al., 2010). (For interpretation of the references to color in this gure legend, the reader is referred to the web version
of this article.)
obtained from SuSy-overexpressing potato plants whose leaves accumulate higher ADPG and starch contents than WT leaves (Muoz et al.,
2005). Further endeavors based on the characterization of mutants totally
lacking SuSy will be necessary to conrm (or refute) the involvement of
SuSy in starch biosynthesis in leaves.
2.2. Starch biosynthesis in heterotrophic organs
Sucrose produced in leaves is imported by the heterotrophic organs
and used as carbon source for energy production and starch synthesis in
the amyloplast. As in the case of leaves, various mechanisms have been
proposed to describe starch biosynthesis in the heterotrophic organs,
which together with the mechanisms of sucrose unloading, will be
presented and discussed below.
2.2.1. Mechanisms of sucrose unloading in heterotrophic organs
Unloading of sucrose arriving from aerial parts of the plant into sink
organs occurs either symplastically (moving via plasmodesmata) or
apoplastically. The route depends on the species, organ or tissue. In
the apoplastic delivery of sucrose to rapidly growing sinks, apoplastic
sucrose that has been released into the extracellular space can be split
by cell wall-bound invertases into glucose and fructose for subsequent
uptake into the cell by hexose transporters. Alternatively, sucrose can
be taken up by plasma membrane-bound sink specic sucrose transporters of non-dividing storage sinks (Sauer, 2007). Sucrose is then
transported via yet-to-be-identied tonoplast sucrose transporters to
the vacuole, which serves as temporary reservoir. Apoplastic sucrose
can also be taken up by a sucrose-induced endocytic process, and
transported to the central vacuole of heterotrophic cells (Etxeberria
et al., 2005a, 2005b; for a review see Etxeberria et al., 2012). Endocytic
uptake of extracellular sucrose and subsequent transport to the vacuole

is not in conict with transport through membrane-bound carriers
given that cell homeostasis can be better maintained if both mechanisms
operate in parallel (Etxeberria et al., 2005a). Consistent with this view,
Baroja-Fernndez et al. (2006) reported that carrier-mediated sucrose
import in starved cells of sycamore (Acer pseudoplatanus L.) plays an important role in the overall sucrosestarch conversion process during the
initial period of sucrose unloading, whereas endocytosis may play an important role in transporting the bulk of sucrose to the vacuole and its
subsequent conversion into starch after prolonged period of sucrose
unloading.
2.2.2. Proposed mechanisms of sucrosestarch conversion in heterotrophic
cells
Sucrose entering the heterotrophic cell by any of the mechanisms
described above must be metabolized to molecules that can be
transported to the amyloplast for subsequent conversion into starch.
Because both chloroplasts and amyloplasts are ontogenically related,
and because chloroplasts possess a very active triose-P translocator
connecting the plastidial and cytosolic compartments, it was originally
assumed that cytosolic triose-Ps entering the amyloplast by means of
a triose-P translocator act as precursors for starch biosynthesis (Boyer,
1985). However, NMR spectroscopy experiments of starch biogenesis
in wheat and maize grains using 13C NMR (Hatzfeld and Stitt, 1990;
Keeling et al., 1988) demonstrated that a C-6 molecule, not a triose-P
(C-3), is the precursor of starch biosynthesis in amyloplasts. Further investigations of the enzyme capacities of amyloplasts (Entwistle and ap
Rees, 1988; Frehner et al., 1990) supported the view that C-6 molecules
entering amyloplasts are the precursors of starch biosynthesis.
Depending on the nature of the C-6 molecule entering the amyloplast
(G6P or ADPG), and depending on the enzymes involved in ADPG

synthesis, various mechanisms have been proposed to explain the
sucrosestarch conversion process in heterotrophic cells.
2.2.2.1. Models of sucrosestarch conversion in heterotrophic cells
according to which AGP is the sole source of ADPG linked to starch
biosynthesis. It is widely assumed that AGP is the sole source of ADPG
in heterotrophic organs of mono- and dicotyledonous plants. Maximal
in vitro AGP activity greatly exceeds the minimum required to support
the normal rate of starch accumulation in starch storing organs
(Denyer et al., 1995; Li et al., 2013; Weber et al., 2000). This would indicate that AGP is not a rate-limiting step in the sucrosestarch conversion process in many heterotrophic organs. In fact, the ux control
coefcient of AGP has been estimated to be as low as 0.08 in some heterotrophic organs (Denyer et al., 1995; Rolletschek et al., 2002; Weber
et al., 2000). As presented above, AGP is a highly regulated enzyme.
However, whereas evidence has been provided that starch synthesis is regulated by post-translational redox modication of AGP
and 3PGA/Pi balance in potato tubers (Tiessen et al., 2003), AGP in developing barley, wheat, pea and bean seeds is insensitive to 3PGA and Pi
regulation (Gmez-Casati and Iglesias, 2002; Hylton and Smith, 1992;

Kleczkowski et al., 1993; Weber et al., 1995).
In heterotrophic organs of dicotyledonous plants, sucrose entering the
cytosolic compartment of the heterotrophic cell is broken down by SuSy
to produce fructose and UDPglucose (UDPG), the latter being converted
to G1P and PPi by UDPG pyrophosphorylase (UGP). G1P is subsequently
metabolized to G6P by means of the cytosolic phosphoglucomutase.
Cytosolic G6P then enters the amyloplast, where it is converted to starch
by the sequential activities of pPGM, AGP and SS (Fig. 5A). Unlike chloroplasts, amyloplasts are unable to photosynthetically generate ATP and
therefore, cytosolic ATP must enter the amyloplast to produce ADPG by
means of AGP. The presence of an ATP/ADP translocator in the envelope
membranes of amyloplasts has been rmly established by different laboratories (Pozueta-Romero et al., 1991b; Tjaden et al., 1998), and evidence
about its relevance in the starch biosynthetic process has been provided
by Tjaden et al. (1998) and Geigenberger et al. (2001) who reported
that potato tubers with decreased plastidic ATP/ADP transporter activities
exhibit reduced starch content.
Genetic evidence demonstrating the importance of SuSy in the
sucrosestarch conversion process in heterotrophic organs of
Starch
10
ADPG
PPi
ATP
ADP
ATP
UDP
16
G1P
ADP
ATP
Carriers/Endocytosis
Sucrose
Sucrose
5
G6P
Fructose
G6P
G1P
GPT
UDPG
UTP PPi
Amyloplast
Cytosol
Starch
Sucrose
Sucrose
UDP
16
Fructose
10
ADPG
ADPG
PPi
G1P
UDPG
ATP UTP PPi
Amyloplast
Cytosol
Fig. 5. Classic interpretation of the mechanisms of sucrosestarch conversion in (A) heterotrophic cells of dicotyledonous plants and (B) cereal endosperm cells according to which AGP is
the sole source of ADPG linked to starch biosynthesis. Note that in heterotrophic cells of dicotyledonous plants (a) AGP is exclusively localized in the plastid, (b) the cytosolic carbon substrate compound entering the amyloplast for subsequent conversion into starch is G6P, and (c) cytosolic UGP is involved in the sucroseG6P conversion process. Plastidial and cytosolic
G1P pools are likely connected by means of a yet to be identied G1P translocator occurring in amyloplasts (Fettke et al., 2010). Note also that in cereal endosperms cells (a) most of AGP
has a cytosolic localization, and (b) the cytosolic carbon substrate compound entering the amyloplast for subsequent conversion into starch is ADPG. Numbering of enzyme activities is the
same as in legends of Figs. 14.
dicotyledonous plants comes from the reduced levels of starch in potato

tubers and carrot roots exhibiting low SuSy activity (Tang and Sturm,
1999; Zrenner et al., 1995). In addition, genetic evidence showing that
starch biosynthesis in heterotrophic organs involves the incorporation
of G6P and its subsequent conversion into ADPG by means of pPGM
and AGP has been obtained from the characterization of Vicia
narbonensis embryos expressing antisense GPT (Rolletschek et al.,
2007) (these embryos, however, expressed lower levels of starchrelated genes such as AGP and SuSy encoding genes), genetically
engineered pea seeds and potato tubers with reduced pPGM
(Harrison et al., 1998; Tauberger et al., 2000) and from V. narbonensis
seeds and potato tubers with reduced AGP activity (Mller-Rber
et al., 1992; Weber et al., 2000). These organs all exhibit reduced starch
content when compared with their corresponding WT organs.
Using disks from WT and pPGM antisensed potato tubers incubated
with radiolabeled G1P, Fettke et al. (2010) reported that G1P can be efciently taken up by potato tuber parenchyma cells and converted to
starch. Moreover, these authors reported that exogenously added
G1P-dependent starch synthesis was diminished in tuber disks of potato plants with low plastidial starch phosphorylase (pSP) activity. Fettke
et al. (2010) thus proposed that G1P can be taken up by amyloplast by a
yet to be identied transporter to be subsequently used as substrate for
pSP- and/or AGP-mediated starch biosynthesis. However, although this
hypothesis can explain the results obtained using potato tuber disks, it
conicts with previous reports using pSP antisensed potato tubers
showing that, in planta, the lack of pSP did not exert any effect on starch
accumulation (Sonnewald et al., 1995). Furthermore, this hypothesis is
hardly reconcilable with the strong reduction of starch content in tubers
of pPGM antisensed potato tubers, indicating that plastidial G6P is
linked to starch biosynthesis (Tauberger et al., 2000).
Unlike dicotyledonous plants where AGP is exclusively localized in
the plastidial compartment, most of AGP in cereal endosperm cells has
a cytosolic localization (Beckles et al., 2001; Denyer et al., 1996;
Johnson et al., 2003; Kleczkowski, 1996; Thornbjrnsen et al., 1996a,
1996b; Villand and Kleczkowski, 1994). Consistently, most of ADPG has
a cytosolic localization in cereal endosperm cells (Liu and Shannon,
1981; Tiessen et al., 2012). Import studies using amyloplasts isolated
from maize and barley endosperms of WT plants and starch-decient/
ADPG-excess maize Zmbt1 and barley Hvbt1 mutants provided genetic
evidence that Zea mays brittle1 (BT1) (ZmBT1) and Hordeum vulgare
BT1 (HvBT1) are membrane proteins that can incorporate cytosolic
ADPG into the amyloplast (Liu et al., 1992; Mhlmann et al., 1997;
Patron et al., 2004; Shannon et al., 1996, 1998). Furthermore, cell fractionation studies revealed that most of ADPG accumulating in highADPG developing endosperms of the Riso 13 Hvbt1 mutant has a cytosolic localization (Tiessen et al., 2012). The overall data thus indicate
that, in cereal endosperm cells, BT1 proteins facilitate the transfer of
cytosolic ADPG into the amyloplast for starch biosynthesis and are
rate-limiting steps in this process. Based on this evidence, a starch biosynthesis model has been proposed for cereal endosperms wherein the
stepwise reactions of SuSy, UGP and AGP take place in the cytosol to convert sucrose into ADPG, which enters the amyloplast by means of BT1 to
be utilized as glucosyl donor for starch biosynthesis (Fig. 5B) (Denyer
et al., 1996; Villand and Kleczkowski, 1994). Genetic evidence demonstrating the importance of SuSy in the sucrosestarch conversion process
in cereal endosperms comes from QTL analyses in maize endosperms
(Thvenot et al., 2005) and from the reduced levels of starch in maize
seeds exhibiting low SuSy activity (Chourey and Nelson, 1976). Evidence
showing the importance of AGP in starch biosynthesis in cereal endosperms comes from AGP mutants exhibiting reduced starch content
(Johnson et al., 2003; Tsai and Nelson, 1966).
2.2.2.2. Models of sucrosestarch conversion in heterotrophic cells
according to which both SuSy and AGP are involved in the synthesis of
ADPG linked to starch biosynthesis. Sufcient evidence exists to support
the view that heterotrophic organs possess in the cytosol important
sources, other than AGP, of ADPG linked to starch biosynthesis. This hypothesis is compatible with the occurrence in the amyloplast envelope
membranes of ADPG transporters (Naeem et al., 1997; PozuetaRomero et al., 1991b; Shannon et al., 1998), and with the occurrence
in heterotrophic organs of cytosolic ADPG metabolizing enzymes such
as ADPG phosphorylase (Dankert et al., 1964; McCoy et al., 2006;
Murata, 1977) and SuSy. As presented in Section 2.1.2, SuSy is a highly
regulated enzyme that can produce ADPG from sucrose and ADP. It
plays a predominant role in determining both sink strength and
phloem loading, and in the entry of carbon into metabolism in
nonphotosynthetic cells. Maximum in vitro ADPG producing SuSy activity is particularly high in starch storing organs, being comparable to that
of AGP in potato tubers (Baroja-Fernndez et al., 2009) and 34 fold
higher than that of AGP in developing barley and maize endosperms
(Baroja-Fernndez et al., 2003; Li et al., 2013). Furthermore, maximal
ADPG producing SuSy activity in heterotrophic organs such as developing maize endosperm greatly exceeds the minimum required to support
the normal rate of starch accumulation in this organ (Li et al., 2013),
which would indicate that ADPG producing SuSy activity is not a rate
limiting step in the sucrosestarch conversion process in heterotrophic
organs.
Essentially in line with investigations describing the occurrence of
simultaneous synthesis and breakdown of glycogen in heterotrophic
bacteria and animals (Bollen et al., 1998; David et al., 1990; Gaudet
et al., 1992; Guedon et al., 2000; Massillon et al., 1995; Montero et al.,
2011), pulse chase as well as starch pre-loading experiments using isolated amyloplasts and heterotrophic organs have provided evidence
about the occurrence of simultaneous synthesis and breakdown of
starch in non-photosynthetic cells of plants under conditions of active
starch accumulation (Neuhaus et al., 1995; Pozueta-Romero and
Akazawa, 1993; Sweetlove et al., 1996). The occurrence of starch futile
cycling in heterotrophic organs has been further fortied by recent
studies showing that global regulators of storage substance accumulation such as FLOURY ENDOSPERM2 (FLO2) positively co-regulate the
expression of starch synthesis and breakdown genes during starch
accumulation in developing rice seeds (She et al., 2010) (see
Section 4.7), and by the fact that down-regulation of starch breakdown
functions in genetically engineered wheat and rice plants results in
increased grain yield (Hakata et al., 2012; Ral et al., 2012) (see
Section 4.6). Furthermore, starch breakdown enzymatic activities are
high in heterotrophic organs (Li et al., 2013). Consistently, alternative/additional models for the sucrosestarch conversion process in
heterotrophic organs of dicotyledonous and monocotyledonous plants
have been proposed according to which (a) cytosolic enzymes such as
SuSy catalyze directly the de novo production from sucrose of ADPG,
which is subsequently imported into the amyloplast by the action of
an ADPG translocator, and (b) pPGM and AGP play an important role
in the scavenging of the glucose units derived from the starch breakdown (Fig. 6). Accordingly, these models preview that the net rate of
starch accumulation in heterotrophic cells is determined by the balance
between the rates of ADPG synthesis in the cytosol, import of cytosolic
ADPG to the amyloplast, starch synthesis and starch breakdown, and
by the efciency with which starch breakdown products can be recycled
back to starch via the coupled reactions of pPGM and AGP. Also, these
models predict that the recovery toward starch biosynthesis of starch
breakdown products will be decient in pPGM and AGP mutants,
resulting in a parallel decline of starch accumulation.
The occurrence of the sucrosestarch conversion pathway illustrated in Fig. 6A implies that neither UDPG produced by SuSy, nor cytosolic
hexose-Ps derived from the action of UGP on UDPG, are involved in
starch biosynthesis in heterotrophic organs of dicotyledonous plants.
This view is consistent with the unexpected WT starch content phenotype of UGP antisensed potato tubers (Zrenner et al., 1993), and with
the WT G6P and G1P contents of starch-decient SuSy antisensed potato tubers (Baroja-Fernndez et al., 2003). Also, this view is coherent
with the unexpected starch-decient phenotype of transgenic potato
10
Starch
10
Sucrose
Sucrose
Sucrose
Sucrose
16
ADPG
ADPG
Fructose
ADP
15
Glucose
12
GPT
G6P
G1P
Amyloplast
Cytosol
B
Fructose ADP
Starch
10
ADPG
ADPG
PPi
9
16
UDP
PPi
9
16
ATP
Fructose
ATP
15
Glucose
12
GPT
G6P
G1P
Amyloplast
G1P
UDPG
UTP PPi
Cytosol
Fig. 6. Suggested interpretation of the mechanisms of sucrosestarch conversion in (A) heterotrophic cells of dicotyledonous plants and (B) cereal endosperms according to which (a) both
SuSy and AGP are involved in the synthesis of ADPG linked to starch biosynthesis, (b) the cytosolic carbon substrate compound entering the amyloplast for subsequent conversion into
starch is ADPG, (c) pPGM and AGP are involved in the scavenging of starch breakdown products, and (d) the rate of starch accumulation would be the result of the net balance between
starch synthesis and breakdown. Note that in heterotrophic cells of dicotyledonous plants neither cytosolic hexose-phosphates, nor cytosolic UGP is involved in the sucrosestarch conversion process. Note also that in cereal endosperm cells SuSy catalyzes the direct conversion of sucrose into both ADPG and UDPG, the latter being converted to ADPG in the cytosol by the
stepwise reactions of UGP and AGP. This view predicts that in pPGM and AGP mutants the recovery toward starch biosynthesis of the glucose units derived from the starch breakdown will
be decient, resulting in a parallel decline of starch accumulation since starch breakdown products (especially glucose and G6P) will leak out the amyloplast. Numbering of enzyme activities is the same as in legends of Figs. 14.
tubers heterologously expressing in the cytosol either yeast invertase

plus glucokinase (Trethewey et al., 1998) or bacterial sucrose phosphorylase (Trethewey et al., 2001), since the high sucrolytic activity
occurring in these tubers leads to drastic reduction of cytosolic sucrose
levels, thus limiting ADPG-producing SuSy activity.
Genetic evidences demonstrating the signicance of ADPGproducing SuSy in the sucrosestarch conversion process in heterotrophic cells of both mono- and dicotyledonous plants are the same as
those presented in Section 2.2.2.1.
3. Microbial volatiles promote both growth and accumulation of
exceptionally high levels of starch in mono- and
dicotyledonous plants
Microbes synthesize and emit many volatile compounds. Volatile
emissions from rhizobacterial isolates of Bacillus spp. promote growth
in Arabidopsis plants by facilitating nutrient uptake, photosynthesis and
defense responses, and by decreasing glucose sensing and abscisic acid
levels (Ryu et al., 2003, 2004; H. Zhang et al., 2008, 2009). In contrast,
volatiles from Pseudomonas spp., Serratia spp. and Stenotrophomonas
spp., and from some fungal species inhibit growth in Arabidopsis plants
(Splivallo et al., 2007; Tarkka and Piechulla, 2007; Vespermann et al.,
2007). Given the lack of knowledge on how microbial volatiles (MVs) affect reprogramming of carbohydrate metabolism in plants, Ezquer et al.
(2010a) explored the effect on starch metabolism of volatiles released
from different microbial species ranging from Gram-negative and
Gram-positive bacteria to different fungi. Toward this end, the authors
measured the starch and soluble sugar contents in leaves of plants
cultured in the presence or in the absence of adjacent microbial cultures.
Noteworthy, Ezquer et al. (2010a) reported that, in the absence of physical contact between plant and microbe, all microbial species tested
(including plant pathogens and microbes that normally do not interact
with plants) emitted volatiles that promoted enhancement of the intracellular ADPG levels and a rapid accumulation of exceptionally high
levels of starch in leaves of both mono- and dicotyledonous plants. Levels
of starch reached by MV-treated leaves were comparable to those occurring in heterotrophic organs such as tubers and cereal seeds. This phenomenon, designated as MIVOISAP (for MIcrobial VOlatiles Induced
Starch Accumulation Process), was also accompanied by strong promotion of growth. Therefore, MIVOISAP cannot be ascribed to utilization
of surplus photosynthates and/or ATP for starch biosynthesis that would

occur under growth arrest conditions.
Transcriptome, metabolite content and enzyme activity analyses of
potato leaves exposed to volatiles emitted by the plant pathogen
Alternaria alternata revealed that starch over-accumulation was accompanied by enhanced 3PGA/Pi ratio and up-regulation of SuSy, acid invertase inhibitors, SSIII and SSIV, starch branching enzyme, GPT2, and
synthesis of phosphatidylinositol-phosphates implicated in processes
such as endocytosis and vesicle trafc (Ezquer et al., 2010a).
MIVOISAP was also accompanied by down-regulation of acid invertase,
plastidial Trxs, plastidial -amylase and pSP, and proteins involved in
the conversion of plastidial triose-phosphates into cytosolic G6P. No
changes were found neither in the expression levels of AGP encoding
genes nor in the redox status of APS1. Furthermore AGP-antisensed potato leaves accumulated exceptionally high levels of starch in the presence of MVs. The overall data thus indicated that potato MIVOISAP is the
consequence of transcriptionally and post-transcriptionally regulated
metabolic reprogramming involving (a) up-regulation of ADPGproducing SuSy, SSIII and SSIV, and proteins involved in the endocytic
uptake and trafc of sucrose, and (b) down-regulation of acid invertase
and starch breakdown enzymes. During potato MIVOISAP there also occurs a down-regulation of ATP-consuming functions involved in internal amino acid provision such as proteases and enzymes involved in
de novo synthesis of amino acids. Ezquer et al. (2010a, 2010b) thus hypothesized that, under conditions of limited ATP-consuming protein
breakdown occurring during exposure to MVs, the surplus ATP and carbon are diverted from protein metabolism to starch biosynthesis.
Time-course analyses of starch and maltose contents in illuminated
Arabidopsis leaves of plants exposed to volatiles emitted by A. alternata
revealed that both starch synthesis and -amylase-dependent starch
degradation were enhanced upon MV treatment, although the nal
balance was positive for synthesis over breakdown (J. Li et al., 2011).
The increase of starch content in illuminated leaves of MV-treated hy1/
cry1, hy1/cry2 and hy1/cry1/cry2 Arabidopsis mutants was many-fold
lower than that of WT leaves, indicating that Arabidopsis MIVOISAP is
subjected to photoreceptor-mediated control. Transcriptomic analyses
of Arabidopsis leaves exposed to A. alternata volatiles revealed changes
in the expression of genes involved in multiple processes. However, unlike potato, no changes could be observed in the expression of starch related genes (J. Li et al., 2011). Also, unlike potato plants, Arabidopsis
MIVOISAP was accompanied by 2-3-fold increase of the levels of reduced
(active) form of APS1. Using different Arabidopsis knockout mutants J. Li
et al. (2011) observed that the magnitude of the MV-induced starch accumulation was low in mutants impaired in SSIII, SSIV and NTRC. Unlike
WT leaves, no changes in the redox status of AGP occurred in ntrc
mutants, providing evidence that MV-promoted monomerization
(activation) of AGP is mediated by NTRC. The overall data thus strongly
indicated that Arabidopsis MIVOISAP involves a photocontrolled, transcriptionally and post-transcriptionally regulated network wherein photoreceptors and post-translational changes (e.g. changes in the redox
status) of plastidial enzyme(s) such as AGP, SSIII and SSIV play important
roles. Consistent with the possible involvement of changes in redox status of SSs in MIVOISAP, Glaring et al. (2012) have recently reported that
SSI and SSIII are reductively activated enzymes.
4. Biotechnological approaches for increasing starch content in
heterotrophic organs
Despite the monumental progress made in understanding the genetic
and biochemical mechanisms of starch biosynthesis in plants, up to the
late 2000s relatively little had been achieved in terms of increasing starch
content and yield using biotechnological approaches, particularly in heterotrophic organs of cereal crops (Smith, 2008). However, within the last
5 years, a series of major developments has produced signicant results
that indicate the likelihood of enhancing starch production. We next
assess the progress made in molecular strategies, both successful and
11
unsuccessful, for improving starch accumulation in heterotrophic organs

of plants of agronomic interest. Finally we will present some potential
strategies for enhancing starch content.
4.1. Enhancement of AGP activity
Most attempts to increase starch accumulation have focused on enhancing AGP activity. The focus on AGP arose originally from the belief
that this enzymatic reaction catalyzes a rate-limiting step in the process of starch synthesis and thus, increases in its activity would concomitantly result in enhanced starch accumulation.
One strategy for enhancing AGP activity in plants consisted of heterologously expressing glgC16 (a mutant variant of the E. coli glgC gene)
that encodes an allosterically insensitive AGP form. Recent studies,
however, have shown that the allosterically sensitive glgC equally
serves the same purpose, since ectopic glgC expression restores WT
starch content in aps1 Arabidopsis mutants (Li et al., 2012). First attempts to increase AGP activity in plants were made in potatoes
through expression of glgC16 under the control of the constitutive 35S
promoter. Using this approach, Stark et al. (1992) reported that when
the product of glgC16 was targeted to plastids, tubers from the transgenic lines accumulated more starch than control tubers, although
there was no correlation between starch levels and AGP activity in different lines. Subsequent attempts to reproduce these results on a different cultivar of potato expressing glgC16 under the control of a tuberspecic promoter did not render tubers with increased levels of starch
and ADPG, although they showed increased levels of AGP activity
(Sweetlove et al., 1996). Ectopic expression of allosterically insensitive
E. coli AGP has also been used in other crops such as rice (Nagai et al.,
2009; Sakulsingharoj et al., 2004), maize (Wang et al., 2007) or cassava
(Ihemere et al., 2006). In these cases enhanced AGP activity in seeds
resulted in enhanced biomass, but there was no increase in the starch
content on a fresh weight basis.
Other attempts at achieving increased starch content in cereal endosperms have used modied variants of the AGP large subunit, which
contributes little to the catalytic activity of the enzyme but is important
in determining its regulatory properties. Giroux et al. (1996) obtained
maize lines expressing Sh2r6hs (a mutated variant of the AGP large
subunit gene), which encodes an enzyme that is allosterically insensitive. The starch content of these lines was higher than in control lines;
however there was no increase in starch as a percentage of seed weight
and, instead, these lines had signicantly heavier seeds (Giroux et al.,
1996). Ectopic expression of Sh2r6hs has been conducted in maize,
rice and wheat (Meyer et al., 2004; Smidansky et al., 2002, 2003). In
these cases enhanced AGP activity in seeds resulted in increased individual seed weight and seed yield per plant.
Most recently N. Li et al. (2011) have shown that ectopic expression
of either the BT2 gene coding for the small subunit maize AGP, or SH2
gene coding for the maize AGP large subunit, results in enhanced seed
weight and starch content.
4.2. Increasing supply of starch precursors to the amyloplast
As indicated above, synthesis of storage starch in the amyloplast requires the incorporation of ATP and carbon skeletons from the cytosol.
Tjaden et al. (1998) and Geigenberger et al. (2001) provided evidence
that increasing the supply of ATP to the plastid in potato tubers could
be a strategy to enhance tuber starch content, since transgenic potato
tubers ectopically expressing a plastidic ATP/ADP transporter from
Arabidopsis under the control of the cauliower mosaic virus 35S promoter accumulated 2-fold more ADPG and 16%36% more starch per
gram fresh weight than control tubers. Although these results would
suggest that starch content is very sensitive to changes in the transport
machinery that supplies ATP, more recent studies using transgenic potato plants ectopically expressing the Arabidopsis ATP/ADP transporter
under the control of a tuber specic promoter did not result in increased
12
levels of starch (L. Zhang et al., 2008). Furthermore, these studies

showed that increasing G6P import to the amyloplast by ectopic expression of a pea GPT had no effect on tuber starch content or tuber yield. By
contrast, double transformants simultaneously expressing the pea GPT
and the Arabidopsis ATP/ADP transporter exhibited an enhanced tuber
yield and starch content, the overall data indicating that both ATP/ADP
and GPT share control of the starch content and tuber yield, most probably by co-limiting substrate (ATP and hexose-P) supply to AGP.
Indirect evidence that increasing ATP supply to the amyloplast is a
strategy for increasing starch content comes from the studies carried
out by Regierer et al. (2002) who reported that constitutive
downregulation of expression of plastidial adenylate kinase, an enzyme
that catalyzes the inter-conversion of ATP with AMP and ADP, increased
the ADPG and starch contents of potato tubers (see also patent WO 02/
097101 A1). These changes were accompanied by increases in the total
adenylate pools (including ATP), although not by changes in the adenylate energy charge or by changes in developmental phenotype. Regierer
et al. (2002) thus hypothesized that adenylate kinase acts in the ATPconsuming direction, competing for the same ATP pool with AGP. The
increase in starch content (2 fold) is the largest so far reported for any
targeted manipulation of starch synthesis.
4.3. Increasing supply of sucrose to heterotrophic cells
The occurrence of high levels of sucrose transporters in heterotrophic
organs where apoplastic sucrose unloading occurs (e.g. developing rice,
maize, barley, faba bean, and wheat seeds) has led to the conclusion
that sucrose transporters may play a role in sucrose uptake into endosperm cells for the synthesis of storage compounds during grain lling
(Aoki et al., 1999, 2002, 2003; Harrington et al., 1997; Hirose et al.,
1997; Sivitz et al., 2005; Weschke et al., 2000). Supporting this hypothesis, Scoeld et al. (2002) reported that antisense inhibition of the rice sucrose transporter OsSUT1 leads to impaired grain lling, the nal grain
weight being reduced. Toward the aim of evaluating the potential of improved sucrose uptake capacity on wheat grain quality and yield
Weichert et al. (2010) produced transgenic wheat plants ectopically expressing the barley sucrose transporter HvSUT1 under the control of an
endosperm-specic promoter. The authors reported that, although protein content in transgenic grains was higher than in WT grains, starch
concentration was unchanged.
In very early stages of potato tuber development, i.e. during the elongation phase of stolon growth, apoplasmic sucrose unloading predominates over symplastic unloading. Subsequent tuberization process
involves a switch from apoplastic to symplastic phloem unloading
(Viola et al., 2001). Although this would apparently imply that sucrose
transporters play a minor role in sucrose unloading in developing tubers
and subsequent uptake of sucrose in parenchymatic cells, Khn et al.
(2003) reported that reduction of StSUT1 expression in antisensed potato
plants leads to reduced yield during early stages of tuber development,
indicating that in this phase StSUT1 plays an important role for sugar
transport. To evaluate whether it is possible to improve potato plant performance by overexpressing a sucrose transporter Leggewie et al. (2003)
produced transgenic potato plants heterologously expressing a spinach
sucrose transporter under the control of the 35S promoter. These plants
produced tubers with WT levels of starch content and yield. Thus, the
overall data obtained using both mono- and dicotyledonous crops
would indicate that up-regulation of sucrose transport is not a satisfactory strategy for increasing starch content in heterotrophic organs.
4.4. Enhancement of sucrose breakdown within the heterotrophic cell
Inspired on the widely accepted view that cytosolic hexose-Ps are
precursors for starch biosynthesis in heterotrophic organs of dicotyledonous plants (see Fig. 5A), attempts to direct more carbon into starch
production in potato tubers consisted of heterologously expressing in
the cytosol enzymes such as yeast invertase plus bacterial glucokinase,
bacterial sucrose phosphorylase or fructokinase, that facilitate the conversion of sucrose or sucrose breakdown products (i.e. glucose and fructose) into cytosolic hexose-Ps. However, contrary to expectations,
transgenic tubers expressing in the cytosol bacterial sucrose phosphorylase, fructokinase or yeast invertase plus bacterial glucokinase all
exhibited reduced starch content (Davies et al., 2005; Trethewey et al.,
1998, 2001). Despite extensive research, the mechanisms leading to
these unexpected results remain obscure (see Section 2.2.2.2).
More successful attempts to promote sucrosestarch conversion
have centered on enhancing SuSy activity. The focus on SuSy arose
from the belief that this sucrolytic enzyme is a major determinant of
sink strength and starch accumulation in both mono- and dicotyledonous plants. First attempts to increase SuSy activity were conducted in
potatoes through expression of StSUS4 under the control of the constitutive 35S promoter. Baroja-Fernndez et al. (2009) reported that UDPG,
ADPG and starch contents in SuSy overexpressing tubers were higher
than in WT tubers. Furthermore, starch yield per plant was shown to
be higher in SuSy-overexpressing plants than in WT plants, under
both greenhouse and open eld conditions (Baroja-Fernndez et al.,
2009). High starch SuSy-overexpressing tubers exhibited a trendwise increase of AGP activity. Furthermore, they exhibited reduced
acid invertase activity, which together with previous studies showing
that down-regulation of SuSy is accompanied by a concomitant reduction of starch levels and enhancement of acid invertase activity
(Zrenner et al., 1995) indicates that (a) SuSy, but not acid invertase, is
involved in the sucrosestarch conversion process, (b) SuSy and acid invertase compete for the same substrate (sucrose), and (c) the balance
between SuSy- and acid invertase-mediated sucrolytic pathways is a
major determinant of starch accumulation in potato tubers. Interestingly enough SuSy overexpressing tubers exhibited other characteristics of
agronomic interest such as substantial increase in antioxidant activity
and resistance to enzymatic browning when compared to control tubers. Protection against browning was ascribed to the involvement of
SuSy in the production of UDPG necessary for glycosylation and stabilization of polyphenols (patent WO 2010/055186 A1).
Most recently Li et al. (2013) produced transgenic maize plants ectopically expressing StSUS4 under the control of the constitutive UBI promoter. The ve independent transgenic lines characterized produced seeds
exhibiting higher SuSy activity, higher ADPG content and ca. 10% more
starch than WT seeds at the mature stage. Furthermore, StSUS4 expressing seeds contained a higher amylose/amylopectin balance that WT
seeds. SuSy (and AGP) activity in WT seeds greatly exceeded the
minimum required to support normal rate of starch accumulation in developing maize endosperms (Li et al., 2013). Consequently, SuSy overexpression should not lead to enhancement of starch content. To explain
the observed positive effect of SuSy over-expression on starch content,
the authors argued that the rate of starch accumulation is the result of
the balance between SuSyAGPADPG transporter-SS-mediated starch
synthesis, and amylase- and/or SP-mediated starch breakdown. Therefore, up-regulation of SuSy results in a concomitant increase of the
balance between starch synthesis and breakdown, thereby leading to enhancement of starch accumulation. The enhancement of ADPG content in
SuSy over-expressing lines (also occurring in AGP over-expressing rice
endosperms, Nagai et al., 2009) would indicate that one or more reactions
constrain carbon ux from cytosolic ADPG into starch. Li et al. (2013) thus
hypothesized that the transport of ADPG is perhaps the more important
limiting factor in starch synthesis in SuSy over-expressing maize
endosperms.
The overall data thus indicate that enhancement of SuSy activity
represents a useful strategy for increasing starch accumulation and
yield in heterotrophic organs of both mono- and dicotyledonous crops.
4.5. Enhancement of expression of starch synthase class IV
Five distinct classes of SSs are known in plants: granule-bound SS
(GBSS), which is responsible for the synthesis of amylose, and soluble
SS classes I, II, III, and IV (SSI, SSII, SSIII, and SSIV, respectively), which are
responsible for the synthesis of amylopectin. Using developing wheat
endosperms exposed to SS inactivating temperatures, Keeling et al.
(1993) reported that SSs have a starch biosynthesis ux control coefcient approaching to unity. This and the fact that maximum catalytic activity of SS is close to metabolic ux through starch biosynthesis in
several starch storing organs would indicate that SS is a major site of
regulation of starch synthesis, and therefore, a good target for the
production of genetically engineered high-starch plants. However,
ectopic expression of SS genes has hardly ever been used to increase
the accumulation of starch. This is mainly due to the presence of different classes of SSs in plants interacting with each other in a protein complex and displaying a specic role in the synthesis of the nal
architecture of the starch granule (Hennen-Bierwagen et al., 2008).
Consequently, changes in the expression of a single SS may lead to profound and unpredictable effects not only on starch structure, but also on
starch content, revealing that SS isoforms have specic but interdependent roles in starch polymer synthesis. This problem is well
exemplied by the results obtained using transgenic potato plants expressing the E. coli glycogen synthase (an enzyme that catalyzes the
same reaction as SS) whose tubers accumulated less starch than WT tubers. Furthermore, the starch from the transgenic tubers had altered
structural properties (Shewmaker et al., 1994).
Roldn et al. (2007) reported that SSIV T-DNA mutants accumulate
only one large starch granule in Arabidopsis chloroplasts. In addition,
Szydlowski et al. (2009) reported that Arabidopsis SSIII/SSIV double TDNA mutants accumulate very reduced starch levels, the overall data
indicating that SSIV plays a key role both in the starch granule initiation
process and in starch accumulation, being mandatory to render the regular number of starch granule found in WT plants. That elimination of SSIV
results in both reduction of number of starch granules and starch content
in Arabidopsis leaves suggested that the number of starch granules could
represent a regulatory step in the control of starch accumulation
(D'Hulst and Mrida, 2010). Conrming this hypothesis Gmez-Arjona
et al. (2011) reported that SSIV overexpression results in enhanced levels
of transitory starch in Arabidopsis leaves. Noteworthy, high levels of starch
accumulated in the end of the day were completely mobilized during the
night in SSIV overexpressing plants, allowing them to grow at a higher
rate than WT plants. Gmez-Arjona et al. (2011) also reported that ectopic expression of SSIV results in increased levels of reserve starch and yield
in tubers of transgenic potato plants cultured both under greenhouse and
open eld conditions. Interestingly enough, SSIV overexpression was
accompanied by pleiotropic increase in AGP and SuSy activities, reduction
of acid invertase activity and enhancement of ADPG content. GmezArjona et al. (2011) thus hypothesized that enhanced starch accumulation in SSIV overexpressing tubers would be the result of enhanced
SSIV, AGP and SuSy activities and reduced acid invertase. Further investigations will be necessary to determine whether ectopic expression of SSIV
represents a useful strategy for increasing starch content and yield in
other crops of agronomic interest such as cereal crops.
4.6. Blocking starch breakdown
As discussed above (see Section 2.2.2.2), substantial evidence has
been provided showing the occurrence of simultaneous synthesis and
breakdown of starch in some starch accumulating heterotrophic cells.
It is thus highly conceivable that starch accumulated by heterotrophic
organs with active starch turnover is the result of the net balance between starch synthesis and breakdown (Baroja-Fernndez et al., 2003,
2009; Li et al., 2013; Muoz et al., 2006). Consequently, downregulation of starch breakdown functions should be a viable means of
increasing starch content in heterotrophic organs.
It is generally accepted that -amylase plays a central role in endosperm starch degradation. This activity is high in developing seed endosperms during the process of starch accumulation (Hakata et al., 2012;
Li et al., 2013). On the other hand, genes encoding glucan, water-
13
dikinases (GWD) involved in starch phosphorylation required for amylase mediated starch breakdown are expressed in starch storing organs.
Supporting the above hypothesis that reducing starch breakdown
would be a viable means of increasing starch content in storage organs
(see above), Ral et al. (2012) recently reported that RNAi-mediated
down-regulation of GWD in wheat endosperm under the control of an
endosperm-specic promoter resulted in an increase in grain yield determined by increases in seed weight, tiller number, spikelets per
head and seed number per spike (see also patent WO 2009/067751
A1). These changes were not accompanied by changes in the percentage
of starch per total dried weight, which is a phenomenon comparable to
that occurring in AGP overexpressing plants (see above). The work of
Ral et al. (2012) provided a promising mechanism for enhancing biomass and grain yield in wheat, with potential application to other
crops. Further evidence supporting that down-regulation of starch degradation may contribute to enhance starch content in developing seeds
has been provided by Hakata et al. (2012), who reported that suppression of -amylase genes improves the quality of rice grain when plants
are cultured under conditions of high temperature.
4.7. Altering the expression of global regulators
SnRK1 (for Sucrose non-fermenting-1-related kinase-1) is a serine/
threonine protein kinase that forms a complex with sucrose nonfermenting (SNF1) and AMP-activated protein kinase (AMPK). It acts
as a central integrator of transcription networks in plant stress and energy signaling, and as such, plays a role in regulating metabolism in response to carbon availability (Baena-Gonzlez and Sheen, 2008; BaenaGonzlez et al., 2007).
Upon sensing the energy decit associated with stress or nutrient
deprivation, SnRK1 triggers extensive transcriptional changes that contribute to restoring homeostasis. In general, SnRK1 activates genes involved in nutrient remobilization and represses those involved in
biosynthetic processes and storage, thus providing alternative sources
of energy through catabolism of amino acids and starch during nutrient
starvation. Thus, SnRK1 positively regulates the expression of starch
breakdown genes, since antisense expression of SnRK1 reduces amylase gene expression in cultured wheat and rice embryos during
sugar starvation (Laurie et al., 2003; Lu et al., 2007). Intriguingly, although the attributed role of SnRK1 is to repress functions involved in
biosynthetic processes and storage, SnRK1 has been shown to be necessary for starch synthesis, since (a) it is required for SuSy gene expression
in potato tubers (Debast et al., 2011; Purcell et al., 1998), and (b)
McKibbin et al. (2006) reported that tubers of SnRK1 overexpressing potato plants accumulate up to 30% more starch than control tubers. As
discussed by McKibbin et al. (2006), starch content enhancement
resulting from SnRK1 overexpression can be ascribed to up-regulation
of SuSy and AGP activities occurring in the SnRK1 overexpressing
lines. Although more research using different crop species is necessary
to investigate the role of SnRK1 in the control of starch metabolism,
the work of McKibbin et al. (2006) provided evidence that SnRK1
overexpression represents a useful strategy for increasing starch accumulation and yield in heterotrophic organs.
Genome-wide co-expression analyses conducted to identify candidate regulators for starch biosynthesis in rice revealed that Rice Starch
Regulator1 (RSR1), an APETALA2/ethylene-responsive element binding
protein family transcription factor, negatively co-regulates the expression of many starch synthesis genes including those coding for AGP,
SSs, GBSS, branching enzymes and starch debranching enzymes (Fu
and Xue, 2010). RSR1 highly expresses in rice organs and tissues with
reduced starch content such as roots, seedlings and leaves, and expresses to much lesser extent in developing seed endosperms. Although
starch content and amylose/amylopectin balance in RSR1 overexpressing seeds were shown to be comparable to those of WT seeds,
seeds of rsr1 mutants displayed increased amylose content as a consequence of up-regulation of GBSS expression (Fu and Xue, 2010), had
14
WT starch content and were larger than WT seeds, a typical phenotype

of AGP over-expression (see above). It thus appears that downregulation of RSR1 is a good strategy for increasing starch yield in rice.
She et al. (2010) identied FLO2 as a gene highly expressing in developing rice seeds coincident with starch accumulation whose mutation
results in both reduced rice grain size and reduced amylose/amylopectin
balance. The same authors reported that FLO2 positively co-regulates the
expression of genes coding for many starch synthesis enzymes such as
AGP (AGPL14, AGPS1, AGPS2a and AGPS2b), SuSy (SuSy1 and SuSy2),
SSs (SSIIb, SSIIIb, SSIVa), GBSS and branching enzymes (BE1 and BEIIb),
and enzymes involved in starch breakdown such as -amylase
(Amy3C, Amy3D and Amy3E), isoamylases (ISA1 and ISA2) and
pullulanase. Importantly, FLO2 overexpressing rice lines produce larger
and heavier grains compared with WT plants, providing evidence that
FLO2 overexpression is a good strategy for increasing starch content
and yield in rice (She et al., 2010). Noteworthy, FLO2 belongs to a family
of rice genes whose orthologs widely exist in plants. Provided the function of FLO2 orthologs encoded proteins is similar to that of FLO2, it is
likely that up-regulation of FLO2 orthologs in other plant species may result in enhanced starch content and yield. Further research will be necessary to conrm or refute this hypothesis.
4.8. Enhancing trehalose-6-phosphate content
Trehalose-6-phosphate (T6P) is a sugar signal of emerging signicance that has been suggested to regulate starch biosynthesis via Trxmediated posttranslational redox activation of AGP in response to sucrose
in leaves, reporting on metabolite status between cytosol and chloroplast
(Kolbe et al., 2005; Lunn et al., 2006; Schluepmann et al., 2003).
Leaves of transgenic plants with enhanced T6P have increased redox
activation of AGP and increased starch levels, whereas leaves of plants
with reduced T6P show the opposite effect (Kolbe et al., 2005). The occurrence of a positive correlation between intracellular T6P levels,
redox AGP activation and rates of starch synthesis in Arabidopsis (Kolbe
et al., 2005; Lunn et al., 2006), and the promotion of SnRK1-dependent
redox activation of AGP by trehalose or sucrose feeding to potato tuber
disks (Tiessen et al., 2003) led to the hypothesis that T6P is a signal of sucrose status that promotes starch accumulation in a SnRK1-mediated
process involving redox AGP activation (Geigenberger et al., 2005;
Tiessen et al., 2003). However, this proposal conicts with recent ndings showing that T6P is a strong inhibitor of SnRK1 (Martnez-Barajas
et al., 2011; Y. Zhang et al., 2009) and with the fact that genes normally
induced by SnRK1 are repressed by T6P and vice-versa (Y. Zhang et al.,
2009). To investigate the role of T6P signaling in starch biosynthesis in
heterotrophic organs Debast et al. (2011) generated transgenic potato
plants with high levels of T6P as a consequence of the heterologous expression of E. coli OtsA (which encodes a T6P synthase). They also characterized transgenic potato plants with low levels of T6P as a consequence
of the ectopic expression of E. coli OtsB (which encodes a T6P phosphatase). These authors reported that tubers from transgenic lines with elevated T6P levels displayed reduced starch content. On the other hand,
tubers from lines with reduced T6P accumulated WT starch content,
although they showed a strongly reduced yield as a consequence of
down-regulation of cell proliferation and growth. Transcriptional proling of low T6P tubers revealed that SnRK1 target genes are strongly upregulated, which conrms the inhibitory effect of T6P on SnRK1 activity.
Although Debast et al. (2011) concluded that T6P plays an important role
for potato tuber growth, their results indicated that altering T6P intracellular levels is not a good strategy for increasing starch content in heterotrophic organs of plants.
4.9. Potential strategies for increasing starch content in heterotrophic organs
4.9.1. Over-expression of BT1
As pointed out in Section 2.2.2.1, data on ADPG content in seeds of
Zmbt1 maize and Hvbt1 barley mutants, and on ADPG transport
capacities of amyloplasts isolated from Zmbt1 and Hvbt1 seeds indicated

that, in cereal endosperms, BT1 proteins facilitate the transfer of
cytosolic ADPG into the amyloplast for starch biosynthesis and are
rate-limiting steps in this process. This has led to the proposal that upregulation of BT1 function would be a strategy for increasing starch content in cereal seeds (Chen et al., 2012; Slattery et al., 2000).
BT1 proteins belong to the mitochondrial carrier family (MCF) of
proteins that are involved in the transport of metabolites across the mitochondrial membrane (Haferkamp, 2007; Millar and Heazlewood,
2003). Although MCF proteins are all presumed to be targeted to the mitochondrial inner membrane (Millar and Heazlewood, 2003), some of
them occur in other subcellular compartments. Kirchberger et al.
(2007, 2008) for instance provided evidence that BT1 is exclusively
localized to the inner plastidial envelope membranes of plastids. However, more recent studies have shown that BT1 proteins are dually
targeted to mitochondria and plastids (Bahaji et al., 2011c).
Kirchberger et al. (2008) reported that homozygous Atbt1
Arabidopsis mutants displayed an aberrant growth and sterility phenotype, which was ascribed to impairments in export of newly synthesized adenylates from plastids to the cytosol. However, Bahaji et al.
(2011b) have recently shown that the aberrant growth and sterility
phenotype of Atbt1 mutants could be reverted to WT phenotype by specic delivery of AtBT1 to mitochondria, indicating that AtBT1 may play
crucial roles, other than transport of newly synthesized adenylates
from plastids to the cytosol, that are connected to mitochondrial metabolism. Taking into account that (a) similar to AtBT1, ZmBT1 is dually
targeted to plastids and mitochondria, (b) similar to homozygous
Atbt1 Arabidopsis mutants, homozygous Zmbt1 mutant seeds germinate
poorly and produce plants with low vigor (Mangelsdorf, 1926), (c) mitochondria do not synthesize starch, and (d) there are many low
starch maize mutant seeds that germinate and produce plants that
grow normally, Bahaji et al. (2011a) hypothesized that (a) the aberrant
growth and sterility phenotype of homozygous Zmbt1 mutants would
not be ascribed to starch deciency and/or to reduced ADPG transport
activity in endosperm amyloplasts, and (b) similar to AtBT1, ZmBT1
would be involved in yet to be identied processes occurring in mitochondria, other than ADPG transport, that are important for normal
growth and fertility that indirectly inuence starch accumulation. Needless to say further studies are necessary to conrm or refute the latter
hypothesis and to know whether up-regulation of BT1 constitutes a
valuable strategy for increasing starch content and yields.
4.9.2. Blocking the activity of ADPG breakdown enzymes
An alternative approach to enhancing starch content in crops would
be to reduce the rate of ADPG degradation. Plants possess a widely distributed enzymatic activity, designated as ADPG pyrophosphatase
(AGPP), that catalyzes the hydrolytic breakdown of ADPG to G1P and
AMP (Rodrguez-Lpez et al., 2000). Two different protein entities are
responsible for this activity: Nudix hydrolases and nucleotide
pyrophosphatase/phosphodiesterases (NPP). Nudix hydrolases have
been suggested to act as housecleaning enzymes that prevent accumulation of reactive nucleoside diphosphate derivates, cell signaling
molecules or metabolic intermediates by diverting them to metabolic
pathways in response to biochemical and physiological needs
(McLennan, 2006). AGPPs belonging to this family have been found
both in the plastidial and cytosolic compartments (Muoz et al., 2008;
Olejnik and Kraszewska, 2005). Up-regulation of the plastidial AGPP
Nudix hydrolase in transgenic potato plants results in reduced levels
of starch content in both leaves and tubers, indicating that this enzyme
has access to the pool of ADPG linked to the biosynthesis of both transitory and reserve starch in leaves and tubers, respectively (Muoz et al.,
2008). The control of the intracellular levels of ADPG linked to starch
production likely represents an important, though still poorly investigated point of control of starch metabolism. Continued molecular genetic analysis will be needed to determine whether AGPPs play a
crucial role in preventing metabolic ux toward starch in heterotrophic
organs of plants, and whether down-regulation of the AGPP function

constitutes a good strategy for enhancing starch content and yield in
major crops.
4.9.3. Ectopic expression of SuSy in the plastid
As presented in Section 2.1.2., a sizable pool of sucrose has a plastidial localization (Gerrits et al., 2001; see Section 2.1.2). SuSys from
cyanobacteria are highly specic for ADP and have high afnity for
this nucleotide (Curatti et al., 2000; Figueroa et al., 2013; Porchia et al.,
1999). Thus, a more radical possibility for increasing the starch content
of crop plants would be to express SuSy (especially from cyanobacterial
origin) in the plastid to produce ADPG.
4.9.4. Blocking the synthesis of storage proteins
During development of some heterotrophic organs such as tubers
and seed endosperms, cell division is followed by cell elongation, differentiation and storage. When cell division ceases, a sizable pool of carbon
and ATP is mainly utilized to produce massive amount of storage starch
and proteins. We must emphasize that a similar phenomenon has been
observed in bacterial cultures entering the stationary phase, where ATP
and carbon are diverted from processes required for cell division and
growth to glycogen production (Montero et al., 2009; Rahimpour
et al., 2013).
Storage protein production is expensive in terms of carbon and ATP
costs and it is highly likely that both storage protein and starch biosynthetic processes compete for the same ATP and carbon pools. Therefore,
blocking storage protein synthesis during the latter phase of development could be a potential strategy for increasing starch content in heterotrophic organs. One way to do it would consist in altering the
expression of factors globally controlling the production of storage proteins. This, however, is difcult since it has been reported that the expression of genes involved in storage starch and protein synthesis is
highly coordinated by the same factors (Giroux et al., 1994; She et al.,
2010). One alternative would consist in moderately down-regulating
the expression of genes directly or indirectly involved in the synthesis
of amino acids by using promoters that are specically expressed during
the latter part of the heterotrophic organ development. In this respect
we must emphasize that accumulation of exceptionally high levels of
starch in leaves of plants exposed to MVs was accompanied by reduction of the levels of amino acids as a consequence of down-regulation
of genes directly or indirectly involved in amino acid biosynthesis (see
Section 3, Ezquer et al., 2010a).
5. Conclusions and main challenges
The discrepancies between the different proposed models of
starch biosynthesis in leaves (illustrated in Figs. 14) and heterotrophic organs (Figs. 5 and 6), and the unexpected failures of some molecular strategies aimed at improving storage starch content in
plants denotes that our knowledge of the starch biosynthetic process
is still at a rudimentary level. Better understanding of the regulation
of starch synthesis will require additional research, either at the biochemical, cell biological and molecular levels, which in turn will
allow plant breeders and biotechnologists to advance in their
attempts to increase starch in a predictable way. Additional knowledge on resource allocation within the plant, sucrose uptake in heterotrophic cells, sources of ADPG, mechanisms of ADPG transport
in plastids, interaction of the plant with both biotic and abiotic environmental factors, and metabolic interaction of the plastidial compartment with other cellular compartments will be crucial. In this
last respect we must emphasize that recent studies have demonstrated the occurrence of strong, but unexplored metabolic links between plastids and endomembrane organelles. Thus, Nanjo et al.
(2006) and Kitajima et al. (2009) have shown that -amylase and
NPPs trafc between endoplasmic reticulum-Golgi and the plastidial
compartment. Furthermore, Wang et al. (2013) have recently shown
15
that autophagy contributes to starch degradation in N. benthamiana

leaves, cytosolic starch granule-like structures being sequestered
by autophagic bodies and delivered to vacuoles for their subsequent
breakdown. Needless to say, further studies will be necessary to
evaluate the relevance of such links in the overall starch metabolism.
Although the study of endocytosis in higher plants remained dormant because of the notion that this mechanism would not work
against the high turgor pressure in plant cells, mounting evidence
has been compiled during the last years showing that endocytosis
is essential for many processes in plants (Mller et al., 2007; Samaj
et al., 2005), including carbohydrate metabolism. We now know
that in heterotrophic organs where apoplastic sucrose unloading occurs endocytosis may play an important role in transporting the bulk
of sucrose from the apoplast to the vacuole and its subsequent conversion into starch (see Section 2.2.1). Thus, further investigations
will be necessary to understand the molecular and signaling mechanisms involved in this process.
Starch biosynthesis is a highly regulated process, both at transcriptional and post-transcriptional level that is interconnected with a wide
variety of cellular processes and metabolic pathways. Its regulation involves a complex and an as yet not well dened assemblage of factors
that are adjusted to the physiological status of the cell. However, to
date most studies on genetic regulation of starch metabolism have focused on the effects of single genes on starch biosynthesis, and analyses
of the effect of global regulatory factors on starch metabolism, as well as
systematic studies of the regulatory mechanisms of starch biosynthesis
are still scanty. Evidence has been provided that, similar to the bacterial
system where glycogen synthesis and breakdown genes are coexpressed and co-regulated with genes involved in diverse biological
processes (Montero et al., 2011), starch metabolism genes are coexpressed and coordinated with other functions such as amino acid
and fatty acid biosynthesis (Fu and Xue, 2010; Giroux et al., 1994; She
et al., 2010; Tsai et al., 2009). Systematic analyses-based characterization of the interactome of starch metabolism-related genes and proteins
with genes and proteins involved in other biological processes, and the
identication of global factors controlling the expression of core starch
metabolism functions will be crucially important to rationally design
molecular strategies aimed at enhancing storage starch content in heterotrophic organs of main crops. In this respect, we must emphasize
that genome-wide co-expression analyses, which are based on the assumption that genes with similar expression patterns are more likely
to be functionally related, have proven to be a powerful tool to identify
regulatory factors in transcriptional networks regulating starch metabolism in crop species such as rice and potato (Ferreira et al., 2010; Fu
and Xue, 2010). Similar type of comparative transcriptome and proteome analyses between different plant organs of WT plants and mutants
with altered starch content should be conducted using a wide range of
species of agronomic interest to identify major determinants and global
regulators of starch biosynthesis.
MV-promoted accumulation of exceptionally high levels of starch is
a recently discovered mechanism for the elicitation of plant carbohydrate metabolism by microbes that still needs to be investigated in
more detail before it can be fully understood and used as a source of
ideas and tools for strategies to complement breeding and biotechnology programs aimed at improving crop yields. At short run, further efforts
and research will be necessary to identify the volatiles and sensing
mechanisms involved in MIVOISAP.
Acknowledgments
This research was partially supported by the grants BIO2010-18239
from the Comisin Interministerial de Ciencia y Tecnologa and Fondo
Europeo de Desarrollo Regional (Spain) and IIM010491.RI1 from the
Government of Navarra, and by Iden Biotechnology. M.O. was partly
supported by grant N. ED0007/01/01 Centre of the Region Han for Biotechnological and Agricultural Research.
16
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Starch Synthesis

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JBA-06696; No of Pages 20

Biotechnology Advances xxx (2013) xxxxxx

Contents lists available at SciVerse ScienceDirect

Starch biosynthesis, its regulation and biotechnological approaches to improve

Available online xxxx

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

industrial needs and social demands. However, while much is known

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

irreversible upon hydrolytic breakdown of PPi by plastidial alkaline

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

AGP is allosterically activated by 3PGA, and inhibited by Pi

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

Gerrits et al. (2001) have provided evidence that a sizable pool of

analyses (Baroja-Fernndez et al., 2013). Third, HPLC:MS analyses of

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

warrant starch production and rapid connection of starch metabolism

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

uptake of extracellular sucrose and subsequent transport to the vacuole

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

(G6P or ADPG), and depending on the enzymes involved in ADPG

regulation (Gmez-Casati and Iglesias, 2002; Hylton and Smith, 1992;

ATP UTP PPi

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

dicotyledonous plants comes from the reduced levels of starch in potato

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

tubers heterologously expressing in the cytosol either yeast invertase

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

of surplus photosynthates and/or ATP for starch biosynthesis that would

unsuccessful, for improving starch accumulation in heterotrophic organs

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

levels of starch (L. Zhang et al., 2008). Furthermore, these studies

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

WT starch content and were larger than WT seeds, a typical phenotype

capacities of amyloplasts isolated from Zmbt1 and Hvbt1 seeds indicated

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

organs of plants, and whether down-regulation of the AGPP function

that autophagy contributes to starch degradation in N. benthamiana

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

major determinant of ne regulation of transitory starch accumulation in Arabidopsis

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

A. Bahaji et al. / Biotechnology Advances xxx (2013) xxxxxx

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