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 623and evenly brown snacks, as gelling agents, and in photographic
films, whereas high amylopectin starches are useful in the food
industry (to improve uniformity, stability, and texture) and in
the paper and adhesive industries. Both, conventional breeding
and transgenic approaches have been utilized for modification of
starch properties (specifically amylose, amylopectin, and phos-
phate content; Sene et al. 2000, Kossmann and Lloyd 2000).
However, in contrast to the situation described previously for
yield, the use of natural mutants has been far more prevalent in
this instance.
QTL analyses have recently been adopted for identifying the
genetic factors underlying starch structure. Recombinant inbred
lines were produced from parental maize lines of differing starch
structure, and the amylose, amylopectin, and water-soluble frac-
tions were analyzed. The loci linked to these traits were located
on a genetic map consisting of RFLP (restriction fragment length
polymorphism) markers (Sene et al. 2000). Using this strategy,
candidate genes were identified that influenced starch structure.
It is clear that application of these techniques may facilitate fu-
ture breeding approaches to generate modified starch properties.
In addition to plant breeding approaches the use of transgen-
esis has been of fundamental importance for understanding and
influencing starch structure (see Table 32.2). Potato tubers with
decreased expression of AGPase (M ̈uller-Rober et al. 1992) had ̈
decreased amylose contents, down to about 60% of that found
in wild type, and smaller starch granules (Lloyd et al. 1999b).
The reduction in amylose content is most likely due to the de-
creased levels of ADP-glucose found in these plants, since this
leads to selective restriction of granule-bound versus soluble
SSs, the latter having a higher affinity for ADPGlc (Frydman
and Cardini 1967). Similarly, when the plastidial adenylate sup-
ply was altered by changes in the expression of the amyloplas-
tidial ATP/ADP translocator, not only the starch content, but
also its structural properties were altered (Tjaden et al. 1998,
Geigenberger et al. 2001). Tubers of ATP/ADP-translocator
overexpressing lines had higher levels of ADP-glucose and
starch with higher amylose content, whereas the opposite was
true for antisense lines. Furthermore, microscopic examina-
tion of starch grains revealed that their size in antisense tubers
was considerably decreased (by 50%) in comparison with the
wild type.
Antisensing granule-bound SS I (GBSSI) in potato tubers re-
sulted in a starch that was almost free of amylose (Kuipers et al.
1994). This type of starch has improved paste clarity and stabil-
ity with potential applications in the food and paper industries
(Jobling 2004). In contrast to this, the amylose content of potato
starch could not be increased above a value of 25.5% on over-
expression of GBSS, hinting that this enzyme is not limiting in
amylose production (Flipse et al. 1994, 1996). It has additionally
been demonstrated that amylose can be completely replaced by
a branched material that exhibits properties in between those of
amylose and amylopectin following plastid-targeted expression
of bacterial glycogen synthases (Kortstee et al. 1998).
The roles of the other isoforms of starch synthase (SS) are, if
anything, less clear. A dramatic reduction in the expression of
SSI in potato had absolutely no consequences on starch struc-
ture (Kossmann et al. 1999). The effects of modifying SSII wereobserved in pea seeds mutated at therug5 locus, which encodes
this protein. These mutants display a wrinkled phenotype that is
often caused by decreased embryo starch content. Starch gran-
ules in these plants exhibit a striking alteration in morphology in
that they have a far more irregular shape and exhibit a reduction
in medium chain length glucans coupled to an increase in short-
and long-chain glucans. Despite the dramatic changes observed
in the pea mutant, the down-regulation of SSII in the potato
tuber had only minor effects, including a 50% reduction in the
phosphate content of the starch and a slight increase in short
chain length glucans (Kossmann et al. 1999, Lloyd et al. 1999a).
However, it is worth noting that SSII only contributes 15% of the
total soluble SS activity within the tuber. Finally, a third class of
SS has also been identified and has been reported to be the major
form in potato tubers (Marshall et al. 1996). When this isoform
(SSIII) was downregulated by the creation of transgenic potato
plants, deep fissures were observed in the starch granule under
the electron microscope, most probably due to GBSS making
longer glucan chains in this background (Fulton et al. 2002).
However, the starch was not altered in its amylose content, nor
did it display major differences in chain length distribution of
short chains within the amylopectin (Marshall et al. 1996, Lloyd
et al. 1999a). However, dramatic changes were observed when
the structure of the side chains was studied, with an accumu-
lation of shorter chains observed in the transgenic lines (Lloyd
et al. 1999a). In addition to this, the phosphate content of the
starch was doubled in this transformant (Abel et al. 1996).
Given that some of the changes observed on alteration of
a single isoform of SS were so dramatic, several groups have
looked into the effects of simultaneously modifying the activi-
ties of more than one isoform by the generation of chimeric an-
tisense constructs. When potato tubers were produced in which
the activities of SSII and SSIII were simultaneously repressed
(Edwards et al. 1999, Lloyd et al. 1999b), the effects were not
additive. The amylopectin from these lines was somewhat dif-
ferent from that resulting from inhibition of the single isoforms,
with the double antisense plants exhibiting grossly modified
amylopectin consisting of more short and extra long chains but
fewer medium length chains, leading to a gross alteration in
the structure of the starch granules. In another study, the par-
allel reduction of GBSSI, SSII, and SSIII resulted in starch
with less amylose and shorter amylopectin chains, which con-
ferred additional freeze-thaw stability of starch with respect to
the wild type (Jobling et al. 2002). This is beneficial since it
replaces the necessity for expensive chemical substitution reac-
tions, and in addition, its production may require less energy, as
this starch cooks at much lower temperatures than normal potato
starches.
Simultaneously inhibiting two isoforms of starch-branching
enzyme to below 1% of the wild-type activities resulted in highly
modified starch in potato tubers. In this starch, normal, high
molecular weight amylopectin was absent, whereas the amy-
lose content was increased to levels above 70%, comparable
to that in the highest commercially available maize starches
(Schwall et al. 2000; Table 32.2). There was also a major ef-
fect on starch granule morphology. In addition, the phosphorus
content of the starch was increased more than five fold. This