BLBS102-c32 BLBS102-Simpson March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come
32 Starch Synthesis in the Potato Tuber 619phosphorylase in plants, other than the effect of changes in the
concentrations of inorganic phosphate on the activity of the en-
zyme (Stitt and Steup 1985).
In the second case, hydrolytic degradation of linear glucans
in the plastid can involve the combined four action enzymes:
α-amylase,β-amylase,α-glucosidase and disproportionating
enzyme (D-enzyme, glucan transferase). There is now direct
molecular evidence thatβ-amylase (exoamylase) plays a signif-
icant role in this process (Scheidig et al. 2002). This enzyme
catalyzes the hydrolytic cleavage of maltose from the nonreduc-
ing end of a linear glucan polymer that is larger than maltotriose.
Maltotriose is further metabolized by D-enzyme, producing new
substrate forβ-amylase and releasing glucose (Fig. 32.5).
Recent studies document that most of the carbon that re-
sults from starch degradation leaves the chloroplast in the form
of maltose, providing evidence that hydrolytic degradation is
the major pathway for mobilization of transitory starch (Weise
et al. 2004). Elegant studies withArabidopsismutants confirmed
this interpretation and identified a maltose transporter in the
chloroplast envelope that is essential for starch degradation in
leaves (Niittyl ̈a et al. 2004). The further metabolism of mal-
tose to hexose-phosphates is then performed in the cytosol and
is proposed to involve cytosolic forms of glycosyltransferase
(D-enzyme; Lu and Sharkey 2004, Chia et al. 2004),α-glucan
phosphorylase (Duwenig et al. 1997), and hexokinase, similar
to maltose metabolism in the cytoplasm ofE. coli(Boos and
Shuman 1998). It will be interesting to find the potato homolog
of the maltose transporter and to investigate its role during starch
degradation in tubers.
Despite recent progress in clarifying the route of starch degra-
dation inArabidopsisleaves and potato tubers, the regulation
of this pathway still remains an open question. More informa-
tion is available concerning cereal seeds, where the enzymes
involved in starch hydrolysis have been found to be especially
active during seed germination, when starch is mobilized within
the endosperm, which at this stage of development represents
a nonliving tissue. The most studied enzyme in this specialized
system isα-amylase, which is synthesized in the surrounding
aleurone layer and secreted into the endosperm. This activity
and that ofα-glucosidase increase in response to the high levels
of gibberellins present at germination. A further level of control
of the amylolytic pathway is achieved by the action of specific
disulphide proteins that inhibit bothα-amylase and debranching
enzyme. Thioredoxinhreduces and thereby inactivates these
inhibitor proteins early in germination. Glucose liberated from
starch in this manner is phosphorylated by a hexokinase, before
conversion to sucrose and subsequent transport to the developing
embryo (Beck and Ziegler 1989).MANIPULATION OF STARCH YIELD
In potato tubers, like all crop species, there has been considerable
interest to increase the efficiency of sucrose to starch conversion
and thus to increase starch accumulation by both conventional
plant breeding and genetic manipulation strategies. Traditional
methodology based on the crossing of haploid potato lines and
the establishment of a high density genetic map have allowed theidentification of quantitative trait loci (QTL) for starch content
(Sch ̈afer-Pregl et al. 1998); however, this is outside the scope of
this chapter, and the interested reader is referred to Fernie and
Willmitzer (2001). Transgenic approaches in potato have fo-
cused primarily on the modulation of sucrose import (Leggewie
et al. 2003) and sucrose mobilization (Trethewey et al. 1998)
or the plastidial starch biosynthetic pathway (see Table 32.2);
however, recently more indirect targets have been tested, which
are mostly linked to the supply of energy for starch synthe-
sis (see Tjaden et al. 1998, Jenner et al. 2001, Regierer et al.
2002). To date, the most successful transgenic approaches have
resulted from the overexpression of a bacterial AGPase (Stark
et al. 1991) and theArabidopsisplastidial ATP/ADP translocator
(Tjaden et al. 1998), and the antisense inhibition of a plastidial
adenylate kinase (Regierer et al. 2002) in potato tubers.
The majority of previous attempts to improve the starch yield
of potato tubers concentrated on the expression of a more ef-
ficient pathway of sucrose degradation, consisting of a yeast
invertase, a bacterial glucokinase, and a sucrose phosphorylase
(Trethewey et al. 1998, 2001). However, although the transgen-
ics exhibited decreased levels of sucrose and elevated hexose
phosphates and 3-PGA with respect to wild type, these attempts
failed. Tubers of these plants even contained less starch than the
wild type, but showed higher respiration rates. Recent studies
have shown that as a consequence of the high rates of oxygen
consumption, oxygen tensions fall to almost zero within grow-
ing tubers of these transformants, possibly as a consequence of
the high energy demand of the introduced pathway, and this re-
sults in a dramatic decrease in the cellular energy state (Bologa
et al. 2003, Geigenberger 2003b). This decrease is probably the
major reason for the unexpected observation that starch syn-
thesis decreases in these lines. In general, oxygen can fall to
very low concentrations in developing sink organs like potato
tubers and seeds, even under normal environmental conditions
(Geigenberger 2003b, Vigeolas et al. 2003, van Dongen et al.
2004). The consequences of these low internal oxygen concen-
trations for metabolic events during storage product formation
have been ignored in metabolic engineering strategies. Molecu-
lar approaches to increase internal oxygen concentrations could
provide a novel and exiting route for crop improvement.
Another failed attempt at increasing tuber starch accumulation
was the overexpression of a heterologous sucrose transporter
from spinach under the control of the CaMV 35S promoter
(Leggewie et al. 2003). The rationale behind this attempt was that
it would increase carbon partitioning toward the tuber; however,
in the absence of improved photosynthetic efficiency this was
not the case.
With respect to the plastidial pathway for starch synthesis,
much attention has been focused on AGPase. Analysis of potato
lines exhibiting different levels of reduction of AGPase due
to antisense inhibition have been used to estimate flux control
coefficients for starch synthesis of between 0.3 and 0.55 for
this enzyme (Geigenberger et al. 1999a, Sweetlove et al. 1999),
showing that AGPase is collimating for starch accumulation in
potato tubers. In addition to having significantly reduced starch
content in the tubers, these lines also exhibit very high tuber
sucrose content, produce more but smaller tubers per plant, and