258 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
the reductive activation of AGPase in plastids and
the regulatory network that controls the expression
of genes in the cytosol share some common compo-
nents. How the sugar signal is transferred into the
plastid and leads to redox changes of AGPase is still
unknown, but may involve interaction of specific
thioredoxins with AGPase.
Subcellular analyses of metabolite levels in grow-
ing potato tubers have shown that the reaction cat-
alyzed by AGPase is far from equilibrium in vivo
(Tiessen et al. 2002), and consequently, the flux
through this enzyme is particularly sensitive to regu-
lation by the above-mentioned factors. It is interest-
ing to note that plants contain multiple forms of
AGPase, which is a tetrameric enzyme comprising
two different polypeptides, a small subunit and a
large subunit, both of which are required for full
activity (Preiss 1988). Both subunits are encoded by
multiple genes, which are differentially expressed in
different tissues. Although the precise function of
this differential expression is currently unknown, it
seems likely that these isoforms will differ in their
capacity to bind allosteric regulators. If this is in-
deed the case, then different combinations of small
and large subunits should show different sensitivity
to allosteric regulation—such as those observed in
tissues from cereals.
While there is a wealth of information on the reg-
ulation of the plastidial isoforms of AGPase—the
only isoform present in the potato tuber—very little
is known about cytosolic isoforms in other species.
Several important studies provide evidence that the
ADP-glucose produced in the cytosol can be taken
up by the plastid (Sullivan et al. 1991, Shannon et al.
1998). From these studies and from characterization
of mutants unable to transport ADP-glucose, it would
appear that this is a predominant route for starch
synthesis within maize. Despite these findings, the
physiological significance of cytosolic ADP-glucose
production remains unclear for a range of species.
However, it has been calculated that the AGPase ac-
tivity of the plastid is insufficient to account for the
measured rates of starch synthesis in barley en-
dosperm, suggesting that at least some of the ADP-
glucose required for this process is provided by
cytosolic production (Thorbjornsen et al. 1996).
It is interesting to note that the involvement of the
various isoforms of AGPase in starch biosynthesis
is strictly species dependent, whereas the various
starch-polymerizing activities are ever present and
responsible for the formation of the two different
macromolecular forms of starch, amylose and amy-
lopectin (Fig. 11.3). Starch synthases catalyze the
transfer of the glucosyl moiety from ADP-glucose to
the nonreducing end of an -1,4-glucan and are able
to extend -1,4-glucans in both amylose and amy-
lopectin. There are four different starch synthase
isozymes—three soluble and one that is bound to
the starch granule.
Starch branching enzymes (SBE), meanwhile, are
responsible for the formation of -1,6 branch points
within amylopectin (Fig. 11.3). The precise mecha-
nism by which this is achieved is unknown; howev-
er, it is thought to involve cleavage of a linear -1,4-
linked glucose chain and reattachment of the chain
to form an -1,6 linkage. Two isozymes of starch
branching enzyme, SBEI and SBEII, are present
and differ in specificity. The former preferentially
branches unbranched starch (amylose), while the
latter preferentially branches amylopectin. Further-
more, in vitro studies indicate that SBEII transfers
smaller glucan chains than does SBEI and would
therefore be expected to create a more highly
branched starch (Schwall et al. 2000). The fact that
the developmental expression of these isoforms cor-
relates with the structural properties of starch during
pea embryo development is in keeping with this sug-
gestion (Smith et al. 1997). Apart from that, isoforms
of isoamylase (E.C. 3.2.1.68) might be involved in
debranching starch during its synthesis (Smith et al.
2003).
While the pathways governing starch synthesis
are relatively clear, those associated with starch
degradation remain somewhat controversial (Smith
et al. 2003). The degradation of plastidial starch can
proceed via phosphorolytic or hydrolytic cleavage
mechanisms involving -1-4-glucan phosphorylases
or amylases, respectively. The relative importance of
these different routes of starch degradation has been
a matter of debate for many years. The question is
whether they are, in fact, independent pathways,
since oligosaccharides released by hydrolysis can be
further degraded by amylases or, alternatively, by
phosphorylases. In addition to this, the mechanisms
responsible for the initiation of starch grain degrada-
tion in the plastid remain to be resolved, since starch
grains have been found to be very stable and rela-
tively resistant against enzymatic action in vitro.
More recently, molecular and genetic approaches
have allowed rapid progress in clarifying the route of