260 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
linear glucans by cleaving the -1,6 branch points.
Further metabolism of linear glucans could involve
phosphorolytic or hydrolytic routes. In the first case,
-glucan phosphorylase leads to the phosphorolytic
release of Glc1-P, which can be further metabolized
to triose-P within the chloroplast and subsequently
exported to the cytosol via the triose-P/Pitransloca-
tor. Recent results show that the contribution of -
glucan phosphorylase to plastidial starch degrada-
tion is relatively small. Removal of the plastidial
form of phosphorylase in Arabdopsisdid not affect
starch degradation in leaves of Arabidopsis(Zeeman
et al. 2004) and potato (Sonnewald et al. 1995). It
has been suggested that the phosphorolytic pathway
could be more important to degrading starch under
certain stress conditions (i.e., water stress; Zeeman
et al. 2004). However, no regulatory properties have
been described for glucan phosphorylase in plants,
other than the effect of changes in the concentrations
of inorganic phosphate on the activity of the enzyme
(Stitt and Steup 1985).
In the second case, hydrolytic degradation of
linear glucans in the plastid can involve the com-
bined four action enzymes: -amylase, -amylase,
-glucosidase and disproportionating enzyme (D-
enzyme, glucan transferase). There is now direct mo-
lecular evidence that -amylase (exoamylase) plays
a significant role in this process (Scheidig et al.
2002). This enzyme catalyzes the hydrolytic cleav-
age of maltose from the nonreducing end of a linear
glucan polymer that is larger than maltotriose.
Maltotriose is further metabolized by D-enzyme,
producing new substrate for -amylase and releas-
ing glucose (Fig. 11.5).
Recent studies document that most of the carbon
that results from starch degradation leaves the chloro-
plast 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 with Arabidopsismutants confirmed
this interpretation and identified a maltose trans-
porter in the chloroplast envelope that is essen-
tial for starch degradation in leaves (Niittylä et al.
2004). The further metabolism of maltose to hexose-
phosphates is then performed in the cytosol and is
proposed to involve cytosolic forms of glycosyl-
transferase (D-enzyme; Lu and Sharkey 2004, Chia
et al. 2004), -glucan phosphorylase (Duwenig et al.
1997), and hexokinase, similar to maltose metabo-
lism in the cytoplasm of E. coli(Boos and Shuman
1998). It will be interesting to find the potato hom-
olog of the maltose transporter and to investigate its
role during starch degradation in tubers.
Despite recent progress in clarifying the route of
starch degradation in Arabidopsisleaves and potato
tubers, the regulation of this pathway still remains
an open question. More information is available con-
cerning cereal seeds, where the enzymes involved in
starch hydrolysis have been found to be especially
active during seed germination, when starch is mo-
bilized within the endosperm, which at this stage
of development represents a nonliving tissue. The
most studied enzyme in this specialized system is -
amylase, which is synthesized in the surrounding
aleurone layer and secreted into the endosperm. This
activity and that of -glucosidase increase in re-
sponse to the high levels of gibberellins present at
germination. A further level of control of the amy-
lolytic pathway is achieved by the action of specific
disulphide proteins that inhibit both -amylase and
debranching enzyme. Thioredoxin hreduces 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
YIELDIn potato tubers, like all crop species, there has been
considerable interest to increase the efficiency of su-
crose 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 pota-
to lines and the establishment of a high density
genetic map have allowed the identification of quan-
titative trait loci (QTL) for starch content (Schäfer-
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 ap-
proaches in potato have focused 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
11.2); however, recently more indirect targets have
been tested, which are mostly linked to the supply of
energy for starch synthesis (see Tjaden et al. 1998,
Jenner et al. 2001, Regierer et al. 2002). To date, the