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In two forthcoming articles, Thorbjcrnsen

et al. 1 and Denyer et al. 2 report that the

activity of ADP-glucose pyrophosphorylase

(AGPase), a key enzyme of starch biosynthesis, is located predominantly in the
cytosol rather than the amyloplasts of the
seed endosperm of both barley and maize.
The authors used plastid-enriched preparations, and AGPase activities were quantified in comparison with activities of marker
enzymes. The presence of both the cytosolic
and plastidic AGPases was confirmed by
immunological methods, using specific
antibodies. The two reports represent a significant breakthrough for plant biologists
studying starch formation; they are also of
general interest, because they provide an
example of the value of genetic, molecular
and biochemical approaches, which led to
the interest in the subcellular location of
AGPase in cereals, and eventually helped
to redefine the details of a major metabolic
pathway in plants.

ADP-glucose pyrophosphorylase in
the cytosol
AGPase, a two-gene-encoded enzyme s'4,

catalyses the first committed step in starch

biosynthesis. The ADP-glucose formed provides the glucose that is the building block
of starch structure in subsequent reactions
catalysed by starch synthase and starchbranching enzyme. Mutations and antisense experiments that eliminate most or
all AGPase activity cause a severe reduction in starch content, indicating that the
enzyme is the major (if not the only) route
for generating ADP-glucose for starch synthesis ~ . The reduced activity caused by
the mutations is almost certainly large
enough to account for the reduced rate of
starch biosynthesis and for all the other
effects on the composition and morphology
of organs8'5.
For many years, one of the axioms of the
enzymology of starch formation has been
that AGPase activity is confined exclusively
to the plastid 4~7.Although there appears to
be no doubt that amyloplasts (and other
plastids) contain their own isozyme(s) of
AGPase ~'2'4'7"s, scant if any attention has
been paid to the possibility of ADP-glucose
synthesis also occurring outside the plastids. In their papers, Thorbjcrnsen et al. t
and Denyer et al. 2 report that, in barley
and maize seed endosperm, the cytosolic
isoform of AGPase accounts for at least
85% and 95%, respectively, of the total
activity of this enzyme in unfractionated
extracts. The overall activity of AGPase
is in excess of that required for starch
formation in this tissue, suggesting that
ADP-ghicose may also be required for
processes other than starch biosynthesis.

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The cytosolicand amyloplastic isozymes differ

in the relative size of their subunits 1'2, and
they may also differ in kinetic and regulatory properties9. The cytosolic location of
AGPase is consistent with earlier evidence9-1~
that the amino acid sequences (predicted
from the cDNAs) of both the small and
large subunits of the major isozyme of
AGPase in barley and maize seeds apparently lack any cleavable N-terminal transit
peptides. These peptides are required for
all proteins that are encoded by the nucleus
and targeted to the stroma of plastids.

Transport considerations
I f ADP-glucose is formed in the cytosol of

cereal seeds, this would imply that it must

be transported to amyloplasts by a carrier,
and this is probably distinct from the
translocator involved in the transport of
sugar-phosphate 12'1s. A cDNA ( B t l ) has
been cloned from maize seeds that encodes
a putative ADP-glucose translocator (it
has some homology to a yeast adenylate
translocator) 13.The gene corresponds to the
B t l mutation in maize, which disrupts
starch synthesis in the endosperm. The B t l
gene encodes the most abundant protein in
maize amyloplast membranes, and it is
organelle-specific t4. ADP-glucose accumulates in B t l but not wild-type seeds, probably reflecting the lack of an ADP-glucose
carrier in the amyloplast membrane of the
mutant ~5. Based on its abundance and on
reduced starch deposition in B t l seeds, the
Btl protein must play a very important
role in starch biosynthesis in vivo. In contrast to the mitochondrial adenylate carrier,
the plastid adenylate translocator can
transport AMP and ADP-glucose in addition to ATP and ADP (Ref. 12).

Integration with sucrose metabolism

In nonphotosynthetic tissues, the carbon
backbone for starch formation comes from
the metabolism of sucrose, which is the
main transport form for photosynthetically
reduced carbon from leaves (source tissues)

to storage organs (sink tissues). In most

plants, after transport into the storage cell,
sucrose is degraded to sugar phosphates in
the cytosol. The major mechanism appears
to be the sucrose synthase-catalysed cleavage of sucrose to provide UDP-glucose,
which - through the activity of UDPglucose pyrophosphorylase (UGPase) is metabolized to glucose-l-phosphate,
the substrate for AGPase8,1e,17. Fructose,
the other product of sucrose synthasecatalysed sucrose degradation, can also
be metabolized to glucose-l-phosphate via
a set of cytosolic enzymes4,5,s. Whereas it
has been assumed that hexose-phosphate
needs to be transported to plastids for

Copyright 1996 ElsevierScience Ltd. All rights reserved. 1360 - 1385/96/$15.00

November1996,Vol.1, No. 11

363

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Fig. 1. A putative mechanism for coupling sucrose breakdown [catalysed by sucrose

synthase (SuSy)] with ADP-glucose formation in the cytosol of barley and maize
seed endosperm 2,9,17.In this scheme, the UDP and ATP required for sucrose breakdown and the ADP-glucose pyrophosphorylase (AGPase) reactions, respectively, can
possibly be regenerated by a nucleoside diphosphate kinase-catalysed reaction (not
shown), using UTP and ADP as substrates. Note that, for clarity, the scheme contains
no details of the amyloplastic isoform of AGPase, which is also present in seed endosperm ~'2.Abbreviations: StSy, starch synthase; AT, adenylate translocator; UGPase,
UDP-glucose pyrophosphorylase; P, phosphate; PPi, inorganic pyrophosphate.

ADP-glucose synthesis ~ , the data presented

by Thorbjcrnsen et al. 1 and Denyer et al. 2
suggest that it may also be used by cytosolic AGPase. In such a way, close integration of the reactions of sucrose metabolism
and starch synthesis would occur.
In the scheme described, the activity of
the seed cytosolic UGPase, catalysing the
production of glucose-l-phosphate that is
derived from sucrose, is probably directly
coupled to that of cytosolic AGPase, which
utilizes glucose-l-phosphate for ADP-glucose
formation 2'9'17 (Fig. 1). The ADP-glucose
synthesized by cytosolic AGPase has been
proposed9 to be exported to the amyloplasts
via the ADP-glucose/adenylate carrier,
which is present in a variety of plastids TM.
Assuming a close metabolic coupling between the UGPase- and AGPase-catalysed
reactions, a net UDP-glucose to ADP-glucose
conversion can be achieved without any net
input of inorganic pyrophosphate or nucleoside tri- and diphosphates. The scheme
also underlines that there is no need for
ATP synthesis for starch formation in
amyloplasts; instead, import of ADP-glucose
into the plastids may be countered by
export of ADP, the product of the starch
synthase-catalysed reaction 9 (Fig. 1).

cereal seed endosperm. Organization of

starch synthesis in seeds of highly bred
cereal crops (e.g. barley and maize), cultured under optimal conditions and sheltered from the full force of natural selection,
may differ from that in plants that have not
been selected specifically for high starch
content. However, there is no apparent reason
why the AGPase-catalysed reaction must be
confined to plastids, especially in nonphotosynthetic tissues. The presence of the cytosolic isozyme of AGPase that is metabolically
coupled to the amyloplast membrane-bound
adenylate carrier may actually represent a
specialization of the starch-accumulating
tissue, since the sucrose-derived carbon
entering amyloplasts only serves as a substrate for the starch synthesis pathway.
The discovery of the major isozyme of
AGPase located outside the amyloplasts in
cereal seeds 1'2will certainly contribute to a
better understanding of starch production,
and should also help in integrating data
about mechanisms of sucrose-derived
metabolite fluxes in this tissue.

Future prospects
It remains to be seen whether the model
proposed for barley and maize seeds has
any relevance to other cereal species. Even
less clear is whether the cytosolic isozyme
of AGPase occurs in tissues other than

References
1 Thorbjcrnsen, T., Villand, P., Denyer, IL,
Olsen, O-A.and Smith, A.M. (1996) Distinct
isoforms ofADP-glucosepyrophosphorylase
occur inside and outside the amyloplasts in
barley endosperm, Plant J. 10, 243-250

364

November
1996,Vol, 1, No, 11

Leszek A. Kleczkowski
Dept of Plant Physiology, Ume& University,
901 87 Ume& Sweden

2 Denyer, ~, Dunlap, F., Thorbj0rnsen, T.,

Keeling, P. and Smith, A.M. The major form
of ADP-glucosepyrophosphorylasein maize
(Zea mays L.) endosperm is extra-plastidial,
Plant Physiol. (in press)
3 Nelson, O. and Pan, D. (1995) Starch
synthesis in maize endosperms,Annu. Rev.
Plant Physiol. Plant Mol. Biol. 46, 475-496
4 Preiss, J. (1991) Biologyand molecular
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7 ap Rees, T. (1995)Where do plants make
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pp. 143-155, American Society of Plant

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encodes two different transcripts for the
ADP-glucosepyrophosphorylasesmall
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