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Sweetness and Light:
The role of sucrose
in higher plants
Chris Pollock, Andy Cairns, Joe
G a l l a g h e r, J o h n F a r r a r * , D e r i To m o s *
a n d O l g a K o ro l e v a *
The case of temperate grasses and cereals
6
Sucrose as a signal
7
Sucrose-responsive genes within grasses
7
The problem of compartmentation
9
Conclusions
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I G E R I N N O VA T I O N S
SWEETNESS AND LIGHT:
THE ROLE OF SUCROSE IN HIGHER PLANTS
SWEETNESS AND LIGHT:
THE ROLE OF SUCROSE IN HIGHER PLANTS
C h r i s P o l l o c k , A n d y C a i r n s , J o e G a l l a g h e r, J o h n F a r r a r * , D e r i
To m o s * a n d O l g a K o ro l e v a *
T
o the shopper, sucrose is a cheap commodity,
mechanisms exist to ensure that supply and demand
used in a wide variety of products and subject
remain in balance over longer periods. Sucrose is
to a poor press because of its link to tooth decay
believed to play a central role in these regulatory
and obesity. To plant biologists, sucrose is one of the
mechanisms.
most significant compounds in the biosphere. Not only
is sucrose the major stable product of photosynthesis
The case of temperate grasses and cereals
for most plants, but it is also the form in which most
Any treatment that either stimulates photosynthesis
carbon is transported in phloem vessels from leaves
(such as increasing irradiance) or reduces demand for
photosynthetic products (such as chilling distant sink
organs) will lead to a transient additional accumulation
of sugars within the leaf.
In many plants this
accumulation is as a mixture of sucrose and starch, but
in temperate grasses and cereals, novel sugars are
synthesised. These sugars are polymers of fructose
(fructans) that are based upon a single sucrose molecule
Figure 1.1 Structures of plant fructans. In a and b, fructose
residues are connected at different positions of the molecules. A
fructan molecule can contains up to 50 fructose units.
(Figure 1.1). Fructans can accumulate to reach 50% of
the dry weight of the leaf. The kinetics of this process
are quite characteristic (Figure 1.2). Initially, sucrose
accumulates but, after some hours, the leaves acquire
into sink organs such as roots, flowers, grains and
-F
-S
-N
-I
-K
-DP=4
tubers. Thus, much of the carbon in the food we eat has
spent time as sucrose, before being converted into
starch, lipid, protein or animal products.
Sucrose can also act as a storage carbohydrate and a
substrate for biosynthesis, but increasingly it is
perceived as a regulator, with changes in content linked
to changes in the patterns of gene expression. This
article discusses some of the ways in which sucrose
may act to integrate plant metabolism. For a number of
years, we have known that plants have a sophisticated
0
2
4 6 8 10 12 14 16 18 20
TIME AFTER EXCISION (h)
short-term control system that helps to balance the
capacity to fix carbon with the demands of the nonphotosynthetic organs. To achieve this, both sucrose
and starch are stored temporarily in the leaves as a
buffer. What is now clear is that other regulatory
6
Figure 1.2 Thin layer chromatographic separation of soluble
sugars from Lolium temulentum induced to accumulate
carbohydrate by excising and illuminating mature leaves. Larger
fructans (towards the bottom of the plate) accumulate over time.
F, S, N, I, K, and DP4 are fructose, sucrose, neokestose,
isokestose, kestose and tetrasaccharide.
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I G E R I N N O VA T I O N S
25
Sucrose (mg/g fw)
(a)
15
10
5
0
0.0
2.5
5.0
7.5
10.0 12.5
15
10
5
0
0.0
15.0
12
(b)
20
2.5
5.0
Time (h)
7.5
10.0 12.5
15.0
Time (h)
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
(c)
10
8
6
4
2
0
0.0
5.0
100
150
200
Time (h)
0 24 48 72 96 120 144
Figure 1.3 Changes in message abundance for SAM synthase track changes in leaf sucrose content in leaves of Lolium temulentum. Changes
were induced by excision and illumination (a); excision and feeding exogenous sucrose in the dark (b); or by chilling and rewarming roots and
meristems (c). The arrow marks the onset of rewarming.
the ability to use sucrose as a substrate for fructan
organs such that, for example, root excision rapidly
biosynthesis. The ability to make fructans is associated
slows export from leaves, and defoliation leads to a
with increases in the activity of enzymes that transfer
progressive decline in metabolic activity in roots.
fructose residues from sucrose to the growing fructan
Changes in sucrose content often correlate with these
chains.
Our evidence suggests that the initial
developmental and metabolic changes. As well as
accumulation of sucrose triggers the transcription of
specific alterations in gene expression, altered sucrose
new mRNA and the synthesis of new enzymes. Any
contents have been implicated in changes in both the
environmental perturbation that leads to sucrose
rate of cell division and in the patterns of
accumulation
to
morphogenesis. There is still considerable debate as to
concomitant changes in gene expression. At least some
the exact mechanism by which sucrose may regulate
of these changes cause increased activity of the
gene expression, so the evidence is essentially
enzymes that synthesise fructans.
correlative. Sucrose does, however, possess all the
would,
therefore,
also
lead
SWEETNESS AND LIGHT:
THE ROLE OF SUCROSE IN HIGHER PLANTS
Sucrose (mg/g fw)
20
Sucrose (mg/g fw)
Iger pages No. 3 1999 15
attributes of a long-range signal and there is growing
Sucrose as a signal
belief that it is one of the key factors in maintaining the
Changes in sucrose content in other species are also
tight control between sources and sinks within plants.
associated with changes in gene expression. Increases
in sucrose content are implicated in the up-regulation
Sucrose-responsive genes within grasses
(switch on) of some genes and the down-regulation
One way to understand the mechanism of sucrose
(switch off) of others. Since sucrose is transported
regulation is to identify and sequence sucrose-
freely between plant organs, it is possible that these
responsive genes. Using the detached-leaf system
effects on gene expression may be part of a long-
described above, we have isolated a gene whose
distance signalling system that balances metabolism
expression is extremely sensitive to changes in sucrose
between different organs. The circumstantial evidence
abundance (Figure 1.3). Interestingly, this gene codes
is quite strong. The sucrose concentration in any plant
for an enzyme that is not involved in sugar metabolism,
organ is an integral of the recent patterns of supply and
suggesting that this type of regulation may be more
consumption. Translocation of sucrose is known to
widespread.
respond quickly to changes in the balance between
isolated three genes from grass leaves that are involved
As an alternative approach, we have
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in sucrose metabolism and that are closely related to
maize acid invertase genes. These genes behave in
Invertase 1
different ways in response to high sucrose
concentrations (Figure 1.4); one is up-regulated, one
down-regulated and the other is not affected at all.
Invertase 3
0
3
6
9
12
SUCROSE
0
3
6
9
Currently we are carrying out transformation
12
experiments with these genes in both the sense and
SORBITOL
antisense orientation. The plants obtained will allow us
to assign a physiological function to these genes. The
Figure 1.4 Changes in abundance of message for three invertase
family genes that show differential sensitivity to sucrose.
Messenger RNA was extracted from darkened, detached leaves fed
sucrose or sorbitol (as an osmotically active but metabolically
inert control) for up to 12h.
next step will be to isolate the regulatory portions of
these genes that respond to changes in sucrose
abundance.
SUGAR ASSAY: enzymes are added sequentially (steps A,B,C,D)
to measure glucose, fructose, sucrose and fructan individually
Samples of Sugars or Cell Sap (20 pl)
➞
Buffer pH 7.5 (5 nl)
➞
D-glucose (D-fructose) + ATP ➞ G-6-P (F-6-P) + ADP
A) + Hexokinase
➞
G-6-P + NADP ➞ D-gluconate-6-P + NADPH + H+
+ G6PDH
➞
B) + Phosphoglucose isomerase
F-6P ➞ G-6-P
➞
SINGLE CELL
SAMPLING
AND
ANALYSIS
➞
SWEETNESS AND LIGHT:
THE ROLE OF SUCROSE IN HIGHER PLANTS
Invertase 2
C) + Sucrose Phosphorylase
Sucrose + Pi ➞ G-1-P + D-fructose
➞
D) + ß-Fructosidase (invertase)
Fructans (Sucrose)➞ D-glucose + D-fructose
Microscope slide
Pressure
control
Photometer
Aluminium
ring
Stage
Glass
microcapillary
Fluorescence
microscope
Oil
Oil
Cell sap
Cell
Cell saps
Droplets of
reaction buffer
UV light
Figure 1.5 The measurement of sugars from single plant cells. Samples of cell sap are collected using a fine microcapillary tube. These samples
ae then analysed on microscope slides using a photometer coupled to a fluorescence microscope.
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I G E R I N N O VA T I O N S
Table 1.1 Concentration of soluble sugars in different cell types of barley leaves
induced to accumulate carbohydrate by cooling the roots and stem apex
Tissue
Glucose
Fructose
Sucrose
Fructan
(as hexose units)
Epidermis
5
nd*
7
nd
Mesophyll
35
6
169
194
Bundle Sheath
7
1
96
177
* not detected
The problem of compartmentation
resolution view of the ways in which sucrose regulates
Leaves are far from homogeneous organs. Only 55% of
leaf metabolism.
the cells in a wheat leaf are photosynthetic, with the
Conclusions
remainder associated with the epidermis and
We believe that plants contain a complex web of
vasculature. Even within photosynthetic cells, we can
signalling and regulatory systems that balance resource
often distinguish between true mesophyll and bundle
acquisition and allocation and thus optimise overall
sheath cells that surround the vascular bundles. Thus
performance. The sensing of sucrose abundance and its
whole leaf measurements of metabolites, enzyme
effect on patterns of gene expression is only one of
activities or levels of mRNA could mask major shifts in
these. There is also "cross-talk" between these systems.
metabolism between cell types. To address this, we
For example, changes in carbohydrate and nitrate
developed ways of sampling and measuring sugars in
abundance both affect gene expression, but in opposite
single cells (Figure 1.5). We measured sugars in
directions, such that increased N availability stimulates
epidermal, mesophyll and bundle sheath cells, and
carbon acquisition and vice versa.
compared them with the whole leaf values. The
advantage of such systems would be maximal under
carbohydrate balance varied significantly between
fluctuating and resource-limited environments (i.e.
tissues (Table 1.1). Epidermal cells contained only
natural ones), where success is linked to reproductive
small amounts of sugars and these did not change when
fitness or to persistency. Under cultivation, we use
export was restricted.
By contrast, mesophyll and
good management to ensure that inputs are non-
bundle sheath cells accumulated both sucrose and
limiting. Here, however, success is measured by
fructan, but in different proportions, suggesting that the
productivity. A better understanding of long-distance
metabolic control differed between these tissues.
regulatory systems and how they operate should
However, some 40% of the sucrose within the leaf
highlight opportunities to modify them by selective
could not be accounted for in the single-cell samples.
breeding and/or transgenesis. This will help us to
We believe that this sucrose is within the vasculature,
sustain production under conditions that currently
where it cannot be sampled.
trigger reductions in resource acquisition.
In addition to
SWEETNESS AND LIGHT:
THE ROLE OF SUCROSE IN HIGHER PLANTS
Soluble carbohydrate concentrations (mM) as:
The selective
carbohydrates, we are now able to determine the
abundance of mRNA within our single-cell samples and
our next task is to match up tissue-specific changes in
message
abundance
with
the
whole
Contact: [email protected]
*University College of North Wales, Bangor
tissue
measurements reported above. This will give us a high-
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