207
Molecular determinants of sink strength
Karin Herbers and Uwe Sonnewald∗
The manipulation of sink to source relations has been subject
to extensive plant breeding programs aiming to improve
harvest index and thereby crop yield. The introduction of
molecular and biochemical tools has enabled scientists to
investigate the underlying principles. This has opened up the
fascinating possibility of identifying molecular determinants of
sink strength and to further increase yield on a rational basis.
In the past, transgenic plants with alterations in the activity of
only one putative molecular determinant have been created
and this strategy has not resulted in substantial and reliable
increases in yield. Yet, careful molecular and biochemical
investigations have provided valuable insight about carbon flux
into different metabolic pathways at different stages of sink
development and it has become apparent that this metabolic
channelling needs to be exploited by using stage- and
cell-specific promoters in attempts to increase sink strength.
Addresses
Institut für Pflanzengenetik und Kulturpflanzenforschung,
Corrensstrasse 3, D-06466 Germany;
∗e-mail: [email protected]
Current Opinion in Plant Biology 1998, 1:207–216
http://biomednet.com/elecref/1369526600100207
 Current Biology Ltd ISSN 1369-5266
Abbreviations
AGPase ADP glucose pyrophosphorylase
QTL
Quantitative trait loci
as a consequence, is a product of multiple molecular
determinants.
The problem of assigning specific function to molecular
factors contributing to or determining sink strength is
aggravated by the presence of sink organs with different
functions. Sinks can be divided into utilization and storage
sinks. The former, such as meristems, growing roots
and developing leaves, import photoassimilates mainly
for catabolism to sustain growth and development of the
respective organ. The latter are organs such as growing
tubers, tap roots, and seeds and fruits whose primary
function is to store imported carbohydrate as sugars, starch
or oil. Growth of storage sinks is characterized by at least
two stages — increase in cell number and accumulation of
storage compounds. Thus, molecular processes involved
in determining sink strength may alter dynamically with
development and also differ with the function of the
respective sink tissue.
A further level of complexity is associated with the fact
that sink organs are part of the whole plant which leads
to competition among different sink organs for assimilates
produced in source leaves. The dependence on the rest
of the plant strongly contributes to apparent sink strength
defined as the net accumulation rate of dry matter in
sink organs [3]. The overall complexity of ‘sink strength’
within the whole plant, parameters that determine carbon
partitioning between competing sinks and the effects of
environmental factors such as changing temperatures and
water supply have recently been reviewed [1,4–6].
Introduction
There have been long standing arguments about the
definition of sink strength and its validity as a concept
[1]. Nowadays, it has been widely accepted that sink
strength can be defined as the competitive ability of an
organ to import photoassimilates. This capacity has been
proposed to be a product of both sink size and sink activity
[2]. Ho suggested a more general viewpoint — sink size
should be considered as part of the physical constraint and
sink activity as the physiological constraint on an organ’s
assimilate import capacity [3]. Together, the physical and
physiological constraints are thought to determine the
potential sink strength. It was further proposed that sink
size could possibly be reflected by the number of cells in
that sink, and one aspect of the physical constraint could
thus be the genetic determination of cell number [3].
Sink activity is comprised of three important physiological
features: firstly, the unloading of photoassimilates from the
phloem, post-phloem transport and retrieval by sink cells;
secondly, utilization, mainly by respiration, and thirdly,
storage of imported carbohydrates. Thus, sink strength
can be influenced by diverse metabolic processes and,
Here, we will take a reductionist view by focusing on
the sink organ itself and omitting the rest of the plant.
We will describe recent findings related to the molecular
parameters that might control or contribute to potential
sink strength, not taking into account possible physiological constraints imposed by source and transport capacities.
After a few basic considerations of how sink strength
might be achieved in sink organs, recent approaches of
manipulating sink strength will be discussed. As most of
the work of the past two years has focused on tomato fruits,
potato tubers, seeds of legumes, maize and cereals, this
review has been sectioned accordingly.
Cellular and physiological constraints of
carbohydrate import into sink organs
The basis for sink activity as an important component
of overall sink strength resides in the model of Münch,
which postulates that unloading and loading of the
conducting tissue are mainly driven by concentration
and/or osmotic gradients [7]. The efficiency of unloading
in sink organs should, therefore, be determined by the
208
Physiology and metabolism
capacity to remove the imported sugar from the same pool.
Different possibilities exist: firstly, the removal of sucrose
by chemical alteration, for example by hydrolysis thereby
creating a concentration gradient, or by the building up
of high molecular weight compounds, such as starch
or oil, thereby creating both a concentration and an
osmotic gradient; and secondly, the removal of sucrose
from symplastic or apoplastic pools by compartmentation.
Molecular approaches to understand the role of different
enzymes associated with these processes have been
presented [8]. It is obvious that enzymes involved in
sugar transport and carbohydrate metabolism, in particular
those that initiate sugar breakdown and those that are
important in diverting flux into storage compounds, are
possible molecular contributors to potential sink strength.
The involvement of specific sugar transporting and
sucrose degrading enzymes is strongly dependent on the
unloading pathway which varies with type and, possibly,
with the developmental stage of the sink organ [9•].
In addition to its breakdown, the resynthesis of sucrose
has been discussed to be involved in phloem unloading
in growing potato tubers. This seemingly futile cycle is
driven by the activity of sucrose synthase and sucrose
phosphate synthase (SPS) and is believed to act as a fine
control on sucrose uptake. Thus it is tempting to speculate
that the modulation of SPS activity during the storage
phase in potato tubers might result in a higher flux of
imported carbon into starch.
Sieve element unloading and initial steps in
the utilization of sucrose
Four pathways exist for sieve-element unloading and
post-phloem transport (Figure 1). Unloading of sucrose
may exclusively use plasmodesmata forming a functional
symplasmic route between the phloem and the sink
parenchyma cells (Figure 1). Symplasmic transport is
thought to be the general unloading route in many
sink types, including root apices, expanding leaves and
developing potato tubers [9•,10,11]. Control of symplasmic
unloading could be exerted by changes in the structure
and number of plasmodesmata (physical constraints) or
by alterations in plasmodesmatal conductivities, possibly
mediated by concentration or turgor differences between
connected cells (physiological constraints). Despite intense efforts, no genes involved in the regulation of
assimilate transport via plasmodesmatal conductivity have
been isolated so far. Viral movement proteins, required
for the short-distance movement of viral RNAs via
plasmodesmata, may be used to trap host proteins [12]. As
movement proteins have been localized to plasmodesmata,
and in addition been found to alter carbon metabolsim,
the possibility exists that plant proteins, interacting with
movement proteins, are involved in assimilate transport via
plasmodesmata.
The other three pathways (Figure 1) contain an apoplastic
step either directly at the boundary between the sieve
element/companion cell complex and parenchyma cells or
Figure 1
Suc
(a)
Suc
Suc
Suc
Suc
(b)
Suc
Suc
Suc
(c)
Suc
(d)
Suc
Hex
Hex
se/cc
Hex
Parenchyma
Current Opinion in Plant Biology
Models for the different unloading pathways. (a) Plasmodesmata
forming a functional symplasmic route. (b) Leakage of sucrose
from the sieve element/companion cell complex and uptake of
sucrose into parenchyma cells, mediated by retrieval mechanisms of
sucrose/proton symporters. (c) Endocytosis might be an additional
route for the uptake of sucrose from the apoplastic space. (d)
Sucrose leaked into the apoplast may encounter a cell wall invertase
that hydrolyses the disaccharide into corresponding hexose sugars.
The hexose sugars are then taken up by active hexose/proton
symport into sink parenchyma cells. Circles with arrows represent
active sugar transporters, circles with broken arrows indicate passive
leakage and/or facilitated transport from the sieve element/companion
cell complex. Broken arrows indicate passive leakage from the
parenchyma cells. se/cc, sieve element/companion cell complex. Suc,
sucrose; Hex, hexose.
at the boundaries between different types of parenchyma
cells during post-sieve element transport (for details see
[9]). Sucrose efflux from the sieve element/companion cell
complex is felt to occur by simple or facilitated diffusion
down a concentration gradient. Uptake of sucrose into
parenchyma cells might be mediated by active mechanisms involving sucrose/proton symporters. The sucrose
transporters isolated to date have been immunolocalized
to either companion cells in Plantago major or to sieve
elements in tobacco, potato, and in minor veins of tomato
in source and sink tissues [13,14•]. These data suggest
that the sucrose/proton carriers isolated so far — in addition
to their role in phloem loading — might be involved in
sucrose retrieval along the unloading pathway. Recent
work on the role of pyrophosphate in the phloem gives
circumstantial evidence for an active retrieval mechanism
in roots [15•]. Apical root tips of wild-type plants
contain higher levels of soluble sugars than the more
basal parts, especially under high light conditions. In
transgenic tobacco plants expressing a phloem-specific
pyrophosphatase the accumulation of sugars in the tip
is abolished, whereas sugar levels are slightly elevated
at the base [15•]. This observation suggests that, in the
Molecular determinants of sink strength Herbers and Sonnewald
209
species and the expression of those analyzed has been
localized to sink tissues [17•].
transgenic plants, leakage of assimilates in the basal part of
roots cannot be compensated by active retrieval of sucrose
into the phloem to maintain high levels in root tips.
Depending on the unloading pathway in sink tissues
there are different enzymatic routes for the breakdown
of the incoming sugar (Figure 2). After being imported
into the cytosol of sink cells (Figure 1) sucrose may be
cleaved by either sucrose synthase or neutral invertase. In
general it is assumed that sucrose may pass freely through
the tonoplast membrane allowing sucrose import into the
vacuole where it is hydrolyzed by vacuolar acid invertase.
Hexoses in the cytosol (Figure 1) are either taken up into
the vacuole or phosphorylated for further metabolism.
Despite intense efforts, the proteins involved in sucrose
transport of sink parenchyma cells have not been identified. An intriguing speculation is that endocytosis is
a possible mechanism to import sucrose into sink cells
(Figure 1). Indirect evidence in favour of this assumption
stems from experiments where the fluorescent dye Lucifer
Yellow was introduced to the apoplast of potato stolon
cortex and was detected in the vacuoles of all cells within
the tuber a few hours later whereas no fluorescence was
observed in the cytosol [16].
Approaches to identify molecular
determinants of sink strength
Alternatively, sucrose leaked into the apoplast may
encounter a cell wall invertase that hydrolyzes the disaccharide into its corresponding hexose sugars. The latter
are then taken up by an active hexose/proton symport
into sink parenchyma cells (Figure 1). Several hexose
transporter cDNAs have been isolated from different plant
The influence of the enzymes of carbohydrate metabolism
on sink strength have been studied in various ways and
correlations between parameters of sink strength and
enzyme activities have been established. This has been
performed with cultivars of the same species and more
Figure 2
Sucrose
Sucrose
Invertase
Hexoses
Invertase
Glc + Frc
Susy
Frc + UDPGlc
Cell wall
UGPase
HK
PGI/PGM
H6P
Glc1P
Vacuole
Glycolysis
PGI/PGM
HK
Glc
H6P
Glc1P
AGPase
Starch
ADPGlc
Amyloplast
Cytosol
Current Opinion in Plant Biology
Schematic drawing of carbohydrate metabolism in starch storing tissues. Sucrose in the cytosol may either be taken up into the vacuole,
be hydrolyzed by neutral invertase to corresponding hexoses or be cleaved by sucrose synthase (Susy), forming fructose and UDP-glucose.
Sucrose in the vacuole can be hydrolyzed by soluble acidic invertase. The hexoses are either taken up into the vacuole, or phosphorylated in the
cytosol to enter glycolysis. Glucose and/or its phosphorylated intermediates may also be taken up into amyloplasts where starch is synthesized.
ADPGlc, ADP-glucose; Frc, fructose; Glc, glucose; Glc1P, glucose-1-phosphate; H6P, hexose-6-phosphate; PGI, phosphoglucoisomerase; PGM,
phosphoglucomutase; UDPGlc, UDP-glucose; UGPase, UDP-glucose pyrophosphorylase.
210
Physiology and metabolism
recently with nearly isogenic lines of quantitative trait loci
(QTL) mapping populations differing in sink strength.
A few QTLs for yield seem to map with enzymatic
activities suggested to be involved in sink strength [18•].
Comparisons were also made between carbon flux during
distinct stages of sink development and the corresponding
tissue enzyme activities. A number of mutants, particularly
in maize, deficient in activities of primary metabolism
were characterized with respect to their sink strength.
Transgenic plants with elevated or reduced levels of
enzymes possibly involved in sink strength were created
and analyzed. The most important recent examples of the
different approaches and the findings gotten through them
will be presented in the following sections.
Tomato fruit sink strength
The development of tomato fruits can be divided into
three stages: a slow growing stage lasting approximately
up to 10 days after anthesis; a fast growing stage up
to 40 days after anthesis; and a maturation phase where
no further carbohydrates are imported. During the early
stage, starch is the main carbohydrate stored and it has
been suggested that the level of starch in the first stage
determines the level of hexoses during later stages and the
rate of fruit growth [19]. The starch-accumulating stage
has been associated with symplasmic post-phloem sugar
transport that shifts to apoplasmic transport during the
phase of rapid hexose accumulation [20]. It is assumed
that the switch from symplasmic to apoplasmic transport
involves an extracellular invertase and that the action of
this invertase influences the rate and extent of hexose
storage. To study the effect of acid invertase on sugar
levels and on fruit size the activity of the enzyme has
been suppressed in antisense experiments [21,22]. About
seven weeks after anthesis the reduction of total invertase
activity was most pronounced in the antisense tomato
plants. Three weeks after anthesis an increased ratio
of sucrose/hexose in fruits was already observed. The
accumulation of sucrose observed in the antisense plants
resulted in a 30% decrease in fruit size [22]. The authors
did not comment on total dry matter accumulation or yield
per plant, therefore, the role of the acid invertase in sink
strength cannot be judged from these experiments. It is
apparent, however, that the acid invertase is responsible
for maintaining high hexose concentrations which in turn
might be responsible for cell expansion growth.
A comparative study of two tomato genotypes differing
in fruit hexose content revealed that the major difference
between the two genotypes was not invertase activity but
the rate of active hexose uptake from the apoplast to the
symplast [23•]. The authors discussed the possibilities that
the different uptake rates might be due to different hexose
transporter activities which in turn could be regulated by
the activity of plasma membrane H+-ATPases.
Sucrose synthase activity has been reported to be positively correlated with final fruit size [24], starch content
and tomato fruit relative growth rate [25] suggesting that
this enzyme contributes to sink strength. Correlations
between sucrose synthase activity and starch synthesis
were relatively weak suggesting that other enzymes might
also be involved in determining the rate of starch synthesis
[25]. Recently, a careful investigation of the enzymes of
the sucrose to starch biosynthetic pathway pointed to three
enzymes co-ordinately regulated with sucrose synthase
and potentially limiting in starch synthesis: fructokinase,
ADP glucose pyrophosphorylase (AGPase) and starch
synthase [26•]. The co-ordinate regulation of fructokinase
and sucrose synthase could be important for maintaining
flux to starch because sucrose synthase is inhibited by
fructose. Unfortunately, to our knowledge neither of these
enzymes has been studied in mutant plants. Recently, two
cDNA clones encoding fructokinase have been isolated
from tomato and the expression of one of these cDNA
clones corresponded with the developmental period of
starch accumulation [27].
Potato tuber sink strength
The process of tuberisation represents the differentiation
of a lateral shoot, the stolon, into a storage organ,
the tuber. Tuber initiation involves a shift from stolon
elongation to radial swelling of the sub-apical region.
This process is accompanied by a decline of alkaline and
acidic invertase and an increase of sucrose synthase and
fructokinase activity [28]. Similar results were recently
obtained with an in vitro synchronized tuberisation system
[29]. The rise in sucrose synthase and fructokinase
activities is positively correlated with the onset of starch
and storage protein biosynthesis. Tuberisation is thus
characterised by a switch from an invertase-sucrolytic to
a sucrose synthase-sucrolytic system [28]. The hydrolysis
of sucrose catalyzed by sucrose synthase could be made
irreversible by immediate fructokinase action on the
reaction product fructose. Estimations for rates of in vivo
fructose phosphorylation in potato tuber discs compared
with extractable maximum catalytic fructokinase activity
revealed that fructokinase is highly regulated and could
catalyze a near rate-limiting reaction and thus could
represent a control point for starch synthesis [30].
To evaluate the different roles of invertases and sucrose
synthase with respect to potato tuber sink strength,
transgenic plants were created expressing a sucrose
synthase antisense RNA [31] and either an apoplastic or
a cytosolic yeast-derived invertase [32•]. The reduction
of sucrose synthase in tubers resulted in an inhibition of
starch and storage protein accumulation and a concomitant
decrease in total tuber dry weight proving the assumption
that sucrose synthase is a major determinant of sink
strength in tubers. Surprisingly, the transgenic tubers
exhibited elevated levels of hexoses which was paralleled
by a 40-fold increase in invertase activities [31]. The
fact that the induced invertase activity was not able
to compensate for decreased sucrose synthase activity
Molecular determinants of sink strength Herbers and Sonnewald
211
argues for metabolic channeling of sucrose via the sucrose
synthase dominated pathway into starch.
degradation and starch biosynthesis utilizes a sucrose
synthase mediated pathway requiring pyrophosphate [33].
The expression of a yeast-derived invertase in the cytosol
and apoplast of tubers revealed that size, number and
morphology of tubers can be determined by apoplastic and
cytosolic hexose levels [32•]. Expression of the invertase in
the apoplast of tubers resulted in reduced tuber number,
increased fresh weight and size per tuber and increased
tuber fresh weight per plant. The dry weight, as a
percentage of fresh weight, decreased possibly due to a
reduction in the starch content, whereas the dry weight per
plant was unaltered. In contrast, the cytosolic expression of
invertase resulted in increased tuber number and reduced
fresh weight per tuber. Thus far, it is unclear whether
the impact on sink development is due to altered water
potential by increased levels of hexoses in the apoplast
or cytosol, respectively, and/or whether apoplastic glucose
acts as a signal to induce cell division in the apoplastic
invertase expressing tubers.
In canola embryos, however, sucrose synthase activity was
found to be much lower during early stages when starch is
accumulating, compared with its activity after the switch to
oil deposition [34•]. Because mature canola seeds contain
54% oil and an insignificant amount of starch, whereas
maize kernels contain 66% starch and only 4% oil, it has
been suggested that sucrose synthase activity reflects the
synthesis of the predominant storage product whether it
be starch in grains or oil in oilseeds [34•].
Both invertase expressing lines had reduced levels of
sucrose but elevated levels of glucose indicating that
sucrose was accessible in both compartments which argues
for an apoplastic unloading route in tubers in addition
to the generally accepted symplastic unloading [10]
Interestingly, cytosolic invertase led to accumulation of
hexose-phosphates, increased glycolytic flux and elevated
respiration rates giving additional evidence that hexoses
and hexose-phosphates resulting from the invertase-dominated pathway cannot be used for additional starch synthesis in tubers (Hajirezaei M, Takahata Y, Threthewey
R, Willmitzer L and Sonnewald U, manuscript in preparation). The expression of apoplastic invertase, however,
did not result in elevated hexose-phosphate levels during
the storage phase despite glucose accumulation indicating
that hexoses may not encounter the cytosol in these plants
(Hajirezaei M, Takahata Y, Threthewey R, Willmitzer
L and Sonnewald U, unpublished data). This is an
exciting biochemical argument for an endocytotic uptake
mechanism into the vacuole as suggested by Oparka and
Prior [16].
Despite the presence of excess enzymatic activities and
metabolites, sink strength in both invertase expressing
lines was not increased. It seems that imported carbohydrates need to be channelled specifically in order to
be appropriately utilized. Differential carbohydrate flux in
maize kernels mediated by specific enzymes support this
concept. Developing maize kernels accumulate primarily
starch in the endosperm and oil in the embryo. Statistical
analyses of enzymatic activities in different parts of the
kernel suggested that glucokinase, fructokinase, and phosphofructokinase activities were primarily associated with
oil accumulation, whereas AGPase and sucrose synthase
were associated with starch accumulation. This indicated
that oil biosynthesis utilizes invertase-mediated sucrose
Fibers, seed coat and cotyledons of developing cotton
seeds consist mainly of cellulose, starch, and storage
proteins or oils, respectively. Studies on the differential
expression of sucrose synthase in these different sink
tissues similarly revealed that sucrose synthase did not
play a role in starch synthesis, instead, its major role
was carbon partitioning to fiber cellulose synthesis and to
protein and lipid synthesis in the cotyledons [35]. In this
system again starch does not appear to be a major storage
form of carbohydrates.
Efficient utilization of sucrose for starch synthesis as
a factor for sink strength has been demonstrated in
transgenic potato plants expressing reduced levels of
AGPase [36]. These tubers did not form any starch,
only sucrose. Total tuber dry weight per transgenic plant
reached only 60–70% of total dry weight of a wild-type
plant despite a significant increase of tuber number per
plant. In the reverse approach, Stark et al. [37] tried to
increase sink strength by the heterologous expression of
a mutant Escherichia coli AGPase gene, the product of
which was subject to reduced allosteric control under
the tuber-specific patatin promoter in potato plants (cv.
Russet Burbank). Tubers of transgenic plants contained
on average 35% more starch than control tubers. More
recently, the same construct has been transformed into
potato plants (cv. Prairie [38•]). Transgenic plants had
AGPase activity elevated by 4 to 5-fold with a concomitant
increased flux into starch roughly proportional to the
increase in AGPase activity [39•]. Despite increased flux
into starch steady-state levels of starch in transgenic
tubers were unaltered due to an increase in starch
turnover possibly by amylolytic activity. There is no
obvious explanation for the different results obtained with
potato tubers expressing E. coli AGPase. Diverse control
mechanisms of starch degradation in different potato
cultivars might be speculated. One important message
drawn from the work by Sweetlove et al. [39•] is that
increased flux into starch does not necessarily result in
starch accumulation. The work also shows that there is
no simple way to ascribe molecular determinants of sink
strength to utilization of imported carbohydrate as the
latter is subject to regulation of synthesis and degradation.
212
Physiology and metabolism
The approach to exploit an AGPase enzyme with reduced
or altered allosteric control for strenghtening sink capacity
has recently also been reported for maize [40•]. The
endosperm-specific shrunken 2 (Sh2) gene encoding the
large subunit of AGPase was modified in vivo by the
excision of the tranposable element dissociation (Ds) from
the region believed to be involved in allosteric regulation.
Revertants with additional tyrosine and serine residues
increased seed weight by 11 to 18% without altering the
amount of starch. These revertants displayed reduced
sensitivity to phosphate which is an allosteric inhibitor of
AGPase [40•]. In contrast to the frequent finding that an
increase in seed weight is associated with a reduction in
seed number the revertant plants appeared not to possess
reduced seed numbers and this should lead to increased
yield.
Besides AGPase, soluble starch synthase (SSS) has been
implicated to strongly influence the formation of starch in
wheat grain endosperm [41]. Antisense experiments with
the major form of SSS, however, in potato did not alter
starch content but instead had profound effects on starch
granule morphology [42•,43•], indicating only a minor role
(if any at all) for this isoform of SSS in sink strength, at
least in potato.
Phloem unloading and sucrose breakdown in
seeds
In developing seeds of legumes and cereals, the embryo
is symplastically isolated from the maternal tissue and
assimilates have to pass the apoplastic space before
entering the developing seeds [44]. Tuber and seed sinks
are similar in that both undergo a switch from invertase
dominated to sucrose synthase dominated sucrose cleavage
during early differentiation of the respective sink organ
[28,29,45,46].
Moreover, this switch is accompanied by a shift from
a high hexose to sucrose ratio (the prestorage stage
in seeds) to a stage characterized by a low hexose to
sucrose ratio followed by the accumulation of storage
compounds such as starch, oil and storage proteins (storage
stage). In storage tissues, invertase activity has been
suggested to be correlated with developmental processes
before storage starts [47]. The importance of hexoses
in the development of a sink organ has not only been
demonstrated in potato tubers [32•] and tomato [22] but
also in the invertase-deficient miniature (Mn1) mutant
of maize [48•]. The authors could show that below a
threshold level of 6% wild-type activity the endosperm
specific cell wall invertase controlled the developmental
stability of maternal cells in the pedicel. In Faba bean a
cell wall invertase is expressed during the prestorage phase
in the seed coat where unloading of photoassimilates is
known to take place [45]. It is assumed that hexoses are
taken up by the embryo via a hexose carrier causing a high
hexose/sucrose ratio in the cotyledons [49•]. By comparing
two genotypes of Vicia faba differing in seed fresh weight
it was observed that the large-seeded genotype formed a
higher number of parenchyma cells [50•]. Furthermore,
there was a positive correlation between cell wall invertase
activity, a high hexose/sucrose ratio and mitotic activity
leading to an extended phase of cell division in the large
seeds. Similarly, in canola embryos it has been observed
that acid invertase activity and hexose levels dropped
during the transition from cell division to cell expansion
[34•]. Altogether, the data indicate that invertase activity
possesses the potential to contribute to the physical
constraints of a sink organ.
Cell wall invertase has long been assumed to be associated
with rapidly growing tissues such as utilization sinks (e.g.
meristems, sink leaves). Recently, it has been shown
that an extracellular invertase and a hexose transporter
were induced upon treatment of Chenopodium rubrum cells
with cytokinins known to stimulate cell division [51•].
These results argue for the involvement of apoplastic
unloading of sucrose, degradation by cell wall invertase
with subsequent active hexose uptake in sink cells as one
mechanism by which cytokinin influences cell division.
Conclusions and perspectives
During domestification of crops the ‘improvement’ of
genetic loci was possibly associated with largely ‘optimized’ enzymatic equipment with most enzymes involved
in primary metabolism being in excess. The finding
that control of a pathway is shared by a number of
components [52] and that sink strength is a quantitative
trait [18•] suggests a priori that single enzymatic activities
will not be exclusive molecular determinants of sink
strength. Nevertheless, biochemical investigations during
the last years have pointed to a few enzymes which, due
to their key biochemical role, were considered possible
contributors to sink strength.
It has turned out that the expression of heterologous
enzymes that are not subject to plant regulatory mechanisms are most promising for crop improvement strategies.
Examples are the expression of mutant AGPases in maize
or certain cultivars of potato that lead to increased seed
weight or elevated levels of starch, respectively [37,40•].
Yet, there are several examples where the expression
of enzymes expected to contribute to sink strength did
not result in unequivocal improvements. Two reasons
have been identified to account for these failures: the
intricate regulatory networks of carbohydrate metabolism,
and metabolic channeling. Thus, it has been shown that
an increased rate of starch biosynthesis in potato plants
was accompanied by increased starch degradation resulting
in constant steady-state levels of starch [39•], and that
despite elevated levels of hexose-phosphates in cytosolic
invertase expressing plants these metabolites could not
be used for additional starch biosynthesis (Hazirezaei M,
Takahata Y, Threthewey R, Willmitzer L and Sonnewald
U, unpublished data). In addition, inadequate timing of
Molecular determinants of sink strength Herbers and Sonnewald
transgene expression due to the lack of specific promoters
may also be involved.
The investigations of the past few years have also enlarged
our understanding of how carbon flux is diverted into sink
tissues at different stages of development. For instance,
it has become apparent that sucrolytic pathways can
be dominated either by invertase or sucrose synthase
which, as a consequence, will lead to diverse biochemical
fluxes. As a general finding about the distribution of
invertase and sucrose synthase activities, it can be stated
that high sucrose synthase activity is present in organs
which have acquired their fate as storage sinks. High acid
invertase has often been found in tissues where active cell
elongation and high respiration is occurring. It seems that
hexose accumulation during early developmental stages
may influence the development of a sink organ.
Knowledge about the molecular determinants of the
physical constraints in sink organs is too scarce to judge
their contribution to sink strength. The promotion of
sink strength can be envisioned by additional approaches
that modify higher levels of regulation governing both
the physical and physiological constraints on sink organs.
These could include plant hormones [53] and the
signal transduction pathways regulated by them. Recently,
mutant Arabidopsis plants defective in primary root development (designated pickle) have been described [54•]. It
was shown that the mutation is involved in a gibberellin
signaling pathway which regulates the transition from an
embryonic seedling stage to a differentiated primary root.
Pickle root tips accumulated oil bodies, storage proteins
similar to those observed in seeds, and starch granules. It
is reasonable to assume that the product of the wild-type
pickle gene and related gene products might influence the
development of a sink tissue and/or regulate biochemical
pathways and, therefore, need to be included in any
conceptual designs to improve sink strength and carbon
flux into certain metabolic pathways. Knowledge of such
regulation would also provide the opportunity to suppress
the development of unwanted competing sink organs.
Cloning of QTLs has become tractable due to the
expanding repertoire of molecular mapping and cloning
techniques. QTL analysis combined with advanced backcrossing, in particular including exotic germlines [55•],
might lead to the identification and isolation of genes
important for extending our knowledge about physical
and physiological constraints of sink organs and their
subsequent manipulation in transgenic plants.
Another increasingly important aspect for sink strength
is the role of sugars not only as energy and carbon
sources but as signal molecules in gene expression.
Carbohydrate supply is necessary for the repression of
genes involved in photosynthesis, the glyoxylate cycle and
mobilization of storage compounds, and for the induction
of genes involved in defense and sink functions, i.e. in
213
the synthesis of storage compounds such as starch and
vegetative proteins [56•]. There is increasing evidence that
transduction of the sugar signal(s) to a variety of sugar-regulated genes is mediated by multiple signal-transduction
pathways [57•,58•]. Thus, it has become conceivable to
manipulate sink-specific gene expression independently
from photosynthetic and stress-induced gene expression.
Apoplastic expression of yeast invertase in transgenic
potato plants resulted in enlarged tubers [32•]. This
suggests a specific role for hexoses in cell division during
early development of the respective sink organs. The
transgenic tubers also revealed that invertase expression
during the storage stage of tuber development leads
to wasted carbon flux into respiration instead of starch
(Hazirezaei M, Takahata Y, Threthewey R, Willmitzer
L and Sonnewald U, unpublished data). These results
call for highly specific promoters, such as a promoter
conferring stolon specificity to allow expression of cell wall
invertase protein when the overall cellular state (metabolic
channelling) can comply with it.
Acknowledgements
K Herbers was funded by the European Communities Biotech Programme
as part of the project ‘Biology of Tuber Dormancy and Sprouting’ PL960529.
References and recommended reading
Papers of particular interest published within the annual period of review
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• of special interest
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inhibitor stauroporine. Zymograms revealed a fast and transient induction of
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