13

SourceSink Interaction and Communication in Leaves

Christine H. Foyer
Institute of Grassland and Environmental Research, Aberystwyth, Dyfed, Wales
Nathalie Galtier
Laboratoire du Mtabolisme, Institut National de la Recherche Agronomique
(INRA), Versailles. France
All biomass production depends on photosynthesis. Plants assimilate CO, from
the atmosphere and reduce it to the level of triose phosphates, which can then be
used to produce carbohydrates. principally sucrose and starch. Photosynthetic
carbon assimilation provides the driving force for biomass production, but it is
only one of many factors influencing plant growth and development. There are
many other critical steps, such as sucrose synthesis and transport from the leaf
mesophyll tissues, phloem loading. and carbon partitioning throughout the plant,
that have considerable impact on these processes. In the leaf carbohydrate (starch
and sucrose) synthesis and nitrogen assimilation are the major sinks for the
products of photosynthesis. Precise coupling of these processes allows parallel
responses to changes in environmental, metabolic, and physiological limitations
such as sink demand. The molecular mechanisms whereby these complex
interactions are achieved are far from understood, hut many lines of evidence
suggest that there is metabolic coordination between source and sink reactions that
involves many steps of reciprocal control. Changes in demand result in
modifications in carbohydrate accumulation in the leaf. These may or may not be
associated with the modulation of carbon metabolism. Where photosynthesis is
decreased as a result of sucrose accumulation within the leaf, it does not appear to
result from orthophosphate limitation of photosynthesis but is rather associated
with the regulated decreases in the quantity of photosynthetic apparatus.
I. INTRODUCTION
This review will consider the producerconsumer relationship between source
(net carbon-exporting) and sink (net carbon-importing) tissues and the regulation

of the energy balance that exists between them (Fig. 1). In addition, two important
questions must he addressed. The first is whether it may be possible to achieve
greater crop productivity through more effective photosynthesis. The answer to
this is equivocal: while the ultimate yield of a plant must be related to the net
photosynthesis of the plant over the growing season, the rate of photosynthesis
measured at a given point in lime yields little information concerning ultimate
biomass production. Furthermore, breeding programs selecting for improved
photosynthesis have consistently failed to produce higher yielding varieties
(Evans and Dunstone 1970). The second pertinent question concerning the source
sink interaction is whether photosynthesis responds to the requirements of the
sink for assimilate (Ho 1992) For many years the

hypothesis has prevailed that low sink demand causes a build-up of assimilate in
source leaves and this, in turn, decreases the rate of photosynthesis (King et al.
1971; I lerold 1980; Neales and [neon 1968). "Feedback" regulation or "sink"
regulation of photosynthesis is a fundamental concept in the sourcesink
relationship. While there is evidence to support the existence of this type of
control, two fundamental features must be remembered in any discussion of the
nature of the regulation involved. Firstly, photosynthesis is, more or less,
indispensable to the plant. In addition, any mechanism causing rapid or severe
feedback inhibition would invariably cause photo inhibition, photo oxidation, and
tissue destruction (Foyer et al. 1990). Second, there are many active sinks in
plants that serve to prevent accumulation of assimilate in source leaves. ,
It has become increasingly apparent that the answers to many unresolved
questions concerning the nature of coordinate control in the sourcesink
relationship cannot he obtained by either a purely physiological or purely
biochemical approach. Physiological investigations involving modulation of either
source or sink capacity have failed to yield unequivocal results. It was, therefore,
the objective of several research groups, including our group in Versailles, to use
moleculargenetic manipulations to study the nature of the sourcesink
interaction. Considerable insight was recently provided by the demonstration that
sugars specifically and coordinately repress the expression of several photosynthetic genes (Sheen 1990). This repression by sugars such as sucrose and glucose,
overrides the regulation by light, cell type, and developmental stage. In addition,
the expression of the putative phosphate translocation ceased when detached
leaves were incubated with sucrose in the light (Schulz et al. 1993). The ability to
generate transgenic plants, modified in the activities of key component enzymes,
has provided a powerful tool with which to study the physiological roles of
individual enzymes in carbon metabolism.
Interaction and Communication in Leaves

In any attempt to understand the control of a pathway it is necessary first to

define the pathway in terms of the properties of the enzymes involved and the
metabolic intermediates and their compartmentation. The second step is to
identify the reactions that are close to equilibrium and those that are far removed
from equilibrium in vivo. Before we can assess the mechanisms of coordinate
control it is necessary to identify the regulatory reactions and to assess the
relevant properties of the enzymes of the component pathways in leaves. It is,
thus, important to understand the empirical relationships between thylakoid and
stromal reactions and between CO2 assimilation and carbohydrate synthesis. In
addition, the leaf begins life as a sink organ deriving its sustenance from the
reserves provided by previous photosynthesis. Leaf biogenesis. the development
of the photosynthetic apparatus with the ensuing production of sucrose and the
metabolic requirements that this entails, must be addressed in order to understand
the complex relationships that govern regulation in the mature tissue. In the
immature leaf the mitochondria supply adenosine triphosphate (ATP) for the
energy-dependent reactions of leaf development. The juvenility of tissue requires
import and metabolism of sucrose that is essentially absent from the mature organ.
Sustained high levels of soluble carbohydrates are not compatible with high rates
of photosynthesis since they prevent the synthesis of components of the
photosynthetic apparatus (Baker 1984; Baker and Hardwick 1975; Sheen 1990).
An understanding of the development of photosynthesis and sucrose synthesis,
thus, provides considerable insight into the nature of the subsequent response of
these processes to the requirements of the sink tissues.
II. THE SINK TO SOURCE TRANSITION IN LEAVES
Ontogenic changes in leaf development occur against a background of interaction
with the rest of the plant and changes in environmental conditions (Baker 1984).
The onset of photosynthesis during leaf ontogeny requires the coordination of
numerous events that are modified by both endogenous and environmental
controls. In terms of the present discussion two events are of prime importance to
the sourcesink interaction. These are (1) the development of photosynthetic
capacity and (2) the importexport behavior of the tissues and associated

changes in enzymes of carbohydrate metabolism. The development of

photosynthetic capacity is associated with leaf emergence. Prior to this point the
synthesis of photosynthetic pigments is prevented by the absence of light. Once
the emerging leaf intercepts light, very rapid pigment synthesis ensues. In sugar
beet maximum photosynthetic CO2 assimilation occurred when the leaves had
attained only 22% of their final length (Fellows and Geiger 1974). Although there
is considerable variation between species, maximum photosynthetic capacity
always occurs either before or at the time of full leaf expansion and often before
maximal chlorophyll content has been achieved (Fraser and Bidwell 1974;
Rawson and Constable 1980: Rawson and Hackett 1974).
Very young leaves are not particularly strong sinks. Maximal sink strength and
maximal values for net carbon import occur at a stage of about 10% final leaf
area. This falls to about zero when the leaf has achieved about half to threequarters of its maximal photosynthetic capacity (Swanson and Hoddinott 1978).
This varies considerably between species and according to environmental
conditions and is also dependent on the stage when the leaves emerge in the plant
growth cycle. Early-developing leaves generally have greater sink strength than
leaves developing later in the plant growth cycle.
In mature leaves photosynthate, in the form of sucrose, is exported from the
mesophyll to the sieve elements for transport to actively growing regions of the
plant. This process is termed phloem loathing. Although there has been much
disagreement concerning the nature of this process in vivo, it now appears that
there is no single mechanism common to all species. Both symplastic and
apoplastic pathways occur (Gamalei 1989; Van Bel 1993; Van Bel and Gamalei
1991; see Grusak et al., this volume). Plant species may be categorized according
to the theoretical pathway of phloem loading. The types are (1) symplastic
loading, (2a) as in (1) hut including an apoplastic step in the pathway, (2b) like
(2a) but with transfer cells with highly invaginated cell walls, and (2c) as in type
(2a) but with Kranz anatomy (Gamalei 1989, 1991; Van Bel 1993).
Source leaves may have, to a greater or lesser degree, a futile cycle of sucrose
synthesis and degradation. Huber (1989) observed a correlation between the

sucrose content of leaves and their soluble acid invertase activity. However, in our
experience this correlation did not hold for a large number of species and there is
no clear correlation between the two parameters (Kingston-Smith and Foyer
1996). It was clear, however, that leaves which have a very high soluble acid
invertase activity generally accumulate relatively low amounts of sucrose.
A. Development of Photosynthetic Competence and Associated Modifications
in Sucrose Metabolism
Young growing leaves depend on imported sucrose to feed carbon into
metabolism. Imported sucrose is hydrolyzed to hexose phosphates to supply the
metabolic pathways that support growth and metabolism. A sucrose gradient,
controlled largely by the utilization of sucrose in the cytoplasm of the sink tissues,
sustains phloem unloading into the young leaf. Sucrose is cleaved to the level of
hexoses by two enzymes, (A) invertase (EC 3.2.1.26) that catalyses the reaction
Sucrose glucose + fructose

(1)

and (B) sucrose synthase (SS) (EC 2.4.1.13) that catalyses the reaction
UDP-glucose + fructosesucrose + UDP

(2)

The relative importance of these two enzymes in sucrose cleavage is largely

species-dependent.
There are two enzymes capable of sucrose synthesis in plants. The first is SS
(reaction [2]). However, this enzyme catalyzes a freely reversible reaction. It is
generally found in tissues where sucrose breakdown is predominant (Hawker
1971: ap Rees 1984). The second enzyme implicated in the synthesis of sucrose in
higher plants is sucrose phosphate synthase (SPS) (EC 2.4.1.14), which catalyzes
reaction (3).
UDP-glucose + F6P sucrose 6-phosphate + UDP

(3)

It is this latter enzyme, together with sucrose phosphatase (EC 3.1.3.24), reaction
(4), that is responsible for net sucrose synthesis in vivo.

Sucrose 6-phosphate + H2O Sucrose + Pi

(4)

The overall change in free energy of reactions (3) and (4) strongly favors sucrose
synthesis. The presence of these reactions allows mature green leaves to be net
exporters of photoassimilated carbon.
The developing green leaf must undergo a massive metabolic reorientation in
order to switch from sucrose import to sucrose export. This is related to the
control of gene expression associated with the greening process. High endogenous
sucrose levels, present during the initial period of leaf expansion, can inhibit
chloroplast development. A decrease in sucrose concentration is required in order
to allow the synthesis of the photosynthetic apparatus. In addition, developmental
and environmental controls also influence the morphological modification of the
tissue. During leaf ontogeny potentially photosynthetic leaf cells progressively
acquire photosynthetic capacity by differentiation of proplastids into functional
chloroplasts. The thylakoid system develops and expands in the mesophyll
chloroplasts, and this is followed by accumulation of starch. Finally, the
plastoglobuli form toward the end of the life span of the leaf.
The factors controlling the development of photosynthetic systems in relation to
the control of chloroplast gene expression have been extensively studied
(Gruissem 1989). Active photosystems PS II and I'S I reaction centers start to
accumulate in etiolated leaves very soon after the onset of illumination (Baker
1984; Baker and Butler 1976). Although the relevance of functional studies on
greening of etiolated tissues to an understanding of natural plastid development is
questionable, the early development of the photosystems is supported by
observations on the leaves of monocotyledons such as Zea mays which show
sequential development of chloroplast components and photosynthetic activities
along the leaf (Baker and Leech 1977; Miranda et al. 1981). Maize leaves show a
marked transition of photosynthetic enzyme activities and CO, assimilation
capacity from the leaf base to the tip. Such measurements show that even at the
base of the leaf the photosystems are present and photochemically efficient
(Miranda et al. 1981). The remainder of the photosynthetic apparatus and the

enzymes required for CO, assimilation and net sucrose synthesis are more slow to
develop.
The export of sucrose from the leaf depends on the development of (1)
photosynthetic potential and (2) the pathway of sucrose synthesis. The SPS
activity increases in parallel with the increase in total leaf protein during
development (Walker and Huber 1989). As in fruit development (Hubbard et al.
1991) and in seedling growth (Arai at al. 1991) the transition between sink and
source in leaves is accompanied by a dramatic change in the relative activities of
the enzymes involved in sucrose metabolism (Nguyen-Quoc et al. 1990; Egger
and Hampp 1993). Large changes in the relative activities of invertase, SS, and
SI'S are early events in the transition from net sucrose import to sucrose export.
The ratio of SS to SPS can be used as a measure of the sink-source transition. In
maize leaves and conifer needles (Nguyen-Quoc et al. 1990; Egger and Hampp
1993) the ratio of SS to SPS decreases rapidly during leaf development. The
Inverse relationship between the activities of the respective enzymes of sucrose
synthesis and sucrose degradation is fundamental to the sink to source transition.
In young maize leaves SS activity was very high, but during development it
decreased rapidly mainly by control at the enzyme protein level (Nguyen-Quoc et
al. 1990). This occurred before sucrose synthesis was initiated; SS activity was
almost completely absent from mature maize leaves.
III. RELATIONSHIPS BETWEEN ELECTRON TRANSPORT AND
PHOTOSYNTHETIC

CARBON

ASSIMILATION

IN

SOURCE

LEAVES
Light drives photosynthesis in two ways. It provides the energy that supports
carbon assimilation and carbohydrate synthesis. Second. it establishes the
necessary conditions for activation of the carbon assimilation cycle. The
mechanisms whereby photosynthetic electron transport activity is integrated with
the metabolic activity of the chloroplast are complex and relatively poorly
understood (Foyer et al. 1990). The relationship between these two processes is
essentially a source-sink interaction. The photosynthetic electron transport chain
contains two photosystems (PS II and PS I) that operate sequentially to achieve

the light-driven reduction of nicotinamide-adenine dinucleotide phosphate (NA

DP) with the concomitant production of a proton gradient. The latter is used to
generate ATP. ATP and reduced NADP (NADPH) are consumed during CO 2
assimilation. This producer-consumer relationship between the electron transport
system arid CO, assimilation ensures tight coupling by virtue of the cycling of
intermediates. However, superimposed upon this simple supply and demand
relationship are several levels of sophisticated regulation that prevent harmful
reactions when electron transport rates are decreased in the event of limiting
availability of substrates. High rates of electron transport are difficult to maintain
when the electron acceptor, NADP, and the substrates for photophosphorylation,
ADP and Pi, are not freely available, hut the interception of light energy cannot be
attenuated in the short term. The capacity to regulate the quantum efficiency of the
photosystems and dissipate excess energy as heat is, thus, prerequisite to efficient
photosynthesis.
In the C3 photosynthetic carbon reduction cycle (Benson-Calvin cycle), which is
the primary pathway of carbon assimilation in plants (Fig. 2), several of the
component enzymes are regulated by light (Scheibe 1990). This confers a degree
of flexibility to the CO, assimilation process that is essential for optimizing
resources and preventing build-up of NADPH and ATP that would be detrimental
to the continued function of the electron transport system. Enzyme regulation
may, thus, maintain a thermo

Figure 2 The autocatalytic BensonCalvin cycle showing the three distinct

phases of the cycle: carboxylation, reduction, and generation.
dynamic gradient between the electron transport system and carbon assimilation
by controlling the rate of substrate interconversion in the BensonCalvin. cycle.
In this way it may serve to minimize flux resistances. The activities of at least five
of the enzymes of the BensonCalvin cycle are coordinated with, and respond to,
the capacity of the electron transport system (see Leegood, this volume). Enzymes
such as sedoheptulose-1,7-bisphosphatase (SBPase) and chloroplastic fructose1,6-bisphosphatase (FBPase) are light-activated via the ferredoxinthioredoxin
system (Scheibe 1990). The reactions they catalyze are significantly displaced
from equilibrium. The modulation of the activities of these enzymes controls (1)
the flow of intermediates through the BensonCalvin cycle and (2) carbon flow
toward starch biosynthesis or sucrose synthesis, respectively. Decreases in the
level of the chloroplast form of FBPase caused a decrease in starch accumulation
and an increase in carbon partitioning to sucrose in transgenic potatoes expressing

an F13Pase gene construct in the antisense direction (Kossman et al. 1992).

Superimposed upon these two basic methods of regulation of thylakoid and
stromal reactions are many other facets of control that serve to modulate and
restrain the activity of each individual process so that it is aligned to the capacity
of the other, to light and CO2 availability, and to demand (in terms of triose
phosphate) on the system.
Reduced NADP and ATP are consumed by carbon assimilation in photosynthesis
and carbon oxidation in photorespiration. In both cases the ratio of ATP to
NADPII consumption is the same (about 1.5). For every two electrons that are
derived from water to reduce NADI', one to two molecules of ATI' are formed. It
is widely accepted that a minimum of three protons is required by the ATP
synthetase to synthesize one molecule of ATP from ADP and Pi, but the enzyme
may require four protons per ATP formed (lunge et al. 1970; Rumberg et al.
1990). The number of protons transferred during noncyclic electron flow from
water to NA DP may be either two or three, depending on whether a Q-cycle
operates at the level of the cytochrome b/f complex between the photosystems
(Heber and Walker 1992). Such ratios would, however, not provide sufficient ATP
during noncyclic electron transport to NADI' alone to cover the needs of carbon
assimilation, and additional ATP would be required. The shortfall in protons
required for ATP synthesis could be met by either cyclic electron transport
(Katona et al. 1992) or pseudocyclic electron transport (Schreiber and Neubauer
1990: Foyer and Harbinson 1994).
The rate of electron transfer between the photosystems is relatively stable over a
large range of irradiances (Genty et al. 1989, 1990ab; Harbinson et al. 1990;
Foyer et al. 1992). The clear linear relationship between the quantum efficiency of
photosystem 1 (cDps ,) and that of photosystem 11 el,..
shows that electron transport is largely noncyclic (Harbinson and Foyer 1991). If
some PS l-mediated cyclic electron flow does contribute to ATP synthesis, then its
rate is proportional to the rate of noncyclic electron flow (Harbinson et al. 1990).
The relatively equal distribution of electron transport capacity through both
photosystems suggests that the contribution of cyclic photophosphorylation to
ATP

synthesis

is

relatively

small

and

that

noncyclic

electron

photophosphorylation is the predominant process under most conditions in the

absence of stress.
When electron transport is not limited by the availability of light, the rate
limiting step of the electron transport chain resides in the reaction between
reduced plastoquinone and the cytochrome b/f complex. The rate of this reaction
is determined principally by the concentration of reduced plastoquinone and the
intrathylakoid pH. This reaction determines the capacity for electron transport in
most circumstances.
During the induction period of photosynthesis PS 11 has a very low efficiency
while the l'700 pool and the NA UP system are largely reduced (Foyer et al. 1992).
In this situation photosynthetic electron transport is restricted after PS I.
Activation of the BensonCalvin cycle enzymes alleviates the limitation on
electron transport after PS I. Thereafter, PS I and PS II are modulated in parallel
as would be expected of a system where noncyclic electron flow is predominant.
During the induction phase the activation state of chloroplast FBPase increases in
parallel with PS II, which determines the quantum efficiency of noncyclic electron
flow. This demonstrates the degree of coordination between the capacities of
thylakoid and stromal reactions. There is also a high degree of coordination
between the regulated capacities of the photosystems. When photorespiration is
eliminated by allowing leaves to photosynthesize in air in which the 0 2 content has
been decreased to 2% there is a good correlation between the quantum efficiency
of each photosystem and the quantum yields of CO, assimilation and 0 2 evolution
with increasing irradiance. As irradiance increases, there is a progressive decrease
in the photochemical efficiency of each photosystem.
The measured relationships among 43, and (Pm

iv

and (Pah may provide some

insights into the

responses of the photosynthetic electron transport system to sink limitation
(Harbinson 1994). These changes, however, will only become evident when sink
limitation causes a decreased demand for ATP and NADPH (Pammenter et al.
1993) and this subsequently modifies the regulatory poise of the system. Sink
limitation does not necessarily involve this type of response, particularly if
structural alterations produce changes in the relative amounts of component

proteins by modulation of gene expression (Krapp et al. 1991,1993; Stitt et al.

1990; Schafer et al. 1992). Several groups have shown that as a results of sink
limitation photosynthesis is no longer increased when the oxygen concentration of
the air surrounding the leaves is decreased to 2% (Labate and Leegood 1988;
Sharkey et al. 1986). In the absence of sink limitation the rate of CO 2 assimilation
is increased by up to 40% in a leaf exposed to.2% 0 2, 360 ppm CO2, and 98% N2
compared to the same leaf in air at 25C. Harbinson (1994) has suggested that the
measured relationships among 4>N , (131,s and (1)co2 may provide information on
the responses of the light harvesting and electron transport systems in leaves
subjected to sink limitation.
An index of electron flux through the photosystems (Jps

tt

and jps /) can be

obtained by using the product of the quantum efficiency of the photosystem and
irradiance (Harbinson et al. 1990; Harbinson and Foyer 1991). The activation
states of light-modulated BensonCalvin cycle enzymes increase with the
increase in flux through the photosystems (Harbinson et al. 1990; Foyer et al.
1990) until maximal values are obtained. Measurements of the activity of NADPmalate dehydrogenase (NADP-MDH) suggest that as the flux of electrons through
the photosystems increases, the redox state of the stroma also increases.
Nevertheless, the stroma is kept in a relatively oxidized state (Harbinson et al.
1990). Measurements of NADP-MDH provide an accurate determination of the
reduction state of the stroma. They may have more physiological relevance than
direct measurement of the NADP/NADPH pool. NADP-MDH is localized
exclusively in the cloroplast stroma and subject to modulation by light and the
NADPH/NADP ratio (Scheibe and Stitt 1988; Foyer 1993). The relationship
between the redox state of the NADPH/NADP system and NADP-MDH during
photosynthetic induction is hyperbolic, while the activities of NADP-MDH in
steady-state conditions in leaves are low (Harbinson et al. 1990; Harbinson and
Foyer 1991). These observations suggest that measurements of leaf NADP-MDH
activity in most situations are an accurate reflection of the overall strornal redox
state.

IV. THE PARTITIONING OF CARBON BETWEEN STARCH AND

SUCROSE
In spite of variations in the availability of light and CO 2, photosynthesizing leaves
tend to ensure a continuous day and night export of assimilate to various plant
sinks (Cieger 1987; Ho 1992). A large carbohydrate pool, commonly consisting of
sucrose and starch, is present in leaves. This buffers the variations in the carbon
fixation rate. In general, leaf starch accumulates during the light and is
remobilized at night for sucrose synthesis but patterns of carbohydrate
accumulation vary with external conditions and with plant species.
Photosynthetic sucrose synthesis (Fig. 3) has been reviewed extensively in
recent years (Stitt et al. 1987; Stitt 1989; Stitt and Quick 1989). The activities of
two enzymes, cytosolic FBPase and SPS, are of the same order as the rate of
sucrose synthesis in leaves and much lower than the activities of many .other
enzymes in the pathway. Direct evidence from measurements of the mass action
ratios suggest that these two reactions catalyze nonequilibrium reactions in vivo.
Cytosolic FBPase is strongly inhibited by the regulatory compound fructose-2,6bisphosphate (Fig. 4), while SPS is subject to a complex system of regulation
involving both direct metabolic regulation and covalent modulation of the enzyme
via protein phosphorylation (Doehlert and Huber 1983; Huber et al. 1989; Huber
and Huber 1991). Sucrose phosphate synthase (SI'S) is considered to be
responsive to metabolic regulation by both source and sink. The positive
correlation between the capacity of the leaf to export sucrose and its SPS activity
is particularly striking because other enzyme activities in the leaf show no
correlation to export rate.
Sucrose phosphate synthase is subject to regulation by (1) the metabolic
activator, G6P , and inhibitor, Pi, which bind to the same site on the enzyme, and
(2) covalent modification, involving changes in the phosphorylation state of the
enzyme protein (Fig. 5). Such modulation may follow a circadian rhythm, with
the enzyme being phosphorylated at night and partially dephosphorylated in the
light. The phosphorylated form is less sensitive to activation by G6P and more
inhibited by Pi, while the reverse is true of the dephosphorylated form (KaltTorres and Huber 1987; Stitt et al. 1988). Thus the phosphorylated form is less

active, causing a decrease in SPS activity at night. Both G6P and Pi also influence
the phosphorylation state of SPS. Glucose-6-phosphate (G6P) inhibits
phosphorylation and

Figure 3 The pathways of starch and sucrose synthesis in leaf mcsophyll

cells.
Pi inhibits dephosphorylation (Weiner et al. 1992). This mechanism may explain
the circadian rhythm of SPS activity. As photosynthesis proceeds during the day,
the GP6 concentration in the cytoplasm increases while Pi decreases. This would
lead to dephosphorylation (and activation) of SPS. At night, the inverse would
lead to deactivation of SPS. The level of activation of SPS closely matches the
rate of photosynthesis in spinach leaves (Battistelli et al. 1991). Sucrose
phosphate synthase sensitivity to sucrose and/or G6P levels may provide a
putative mechanism allowing sucrose synthesis to adjust to the rate of sucrose
export (Foyer 1987; Stitt and Quick 1989).

Net starch accumulation is considered to be controlled by the rate of synthesis

but starch degradation may also be regulated (Preiss 1982). The chloroplast
enzyme, ADP-glucose pyrophosphorylase (EC 2.7.7.27), that catalyzes the
interconversion between ADP-glucose and G1P in the reaction
ADP-glucose + PPi G1P + ATP (5)
is considered to play a pivotal role in starch synthesis (Fig. 4). ADP-glucose is the
substrate for the

Figure 4 A scheme for modulation of carbon partitioning involving Pi. Key

regulatory enzymes: (A) ADP-glucose pyrophosphorylase; (B) fructose-1,6bisphosphatase; (C) fructose 6-phosphate kinase; (D) fructose 2,6bisphosphatase, and (E) sucrose phosphate synthase, Stimulatory (+) or
inhibitory () effects of the component metabolites are indicated. Pi,
inorganic phosphate.

starch syntheses and branching enzymes. The reaction is pulled in the

direction of ADP-glucose synthesis by the stromal alkaline pyrophosphatases
(Weiner et al. 1987) that prevent the back reaction to GIP. The possibility of
the existence of an alternative pathway for ADP-glucose synthesis, as
suggested by Pozueta-Romero et al. (1991), is not supported by recent
observations in transformed plants (Okita 1992; Muller-Rober et al. 1992).
Adenosine diphosphateglucose pyrophosphorylase remains the obligate
reaction for ADP-glucose synthesis. Stark et al. (1992) demonstrated that the
amount of starch in plant tissues is regulated by the activity of ADP-glucose
pyrophosphorylase. They showed that transformation with one of the enzyme
subunits in the antisense direction caused the production of plants severely
deficient in ADP-glucose pyrophosphorylase. Such plants were virtually
incapable of starch synthesis. Conversely, plants transformed with a modified
form of the enzyme

Figure 5 Scheme of the regulatory mechanisms involved in modulation of leaf

sucrose phosphate synthase activity.
showed decreased sensitivity to the inhibitor Pi and tended to make much more
starch. The existence of an alternative route for ADP-glucose synthesis, as
suggested by Pozueta-Romero et al. (1991), remains to be demonstrated.

Glycerate 3-phosphate (PGA) is an activator of ADP-glucose pyrophosphorylase,

while Pi is an inhibitor. The synthesis of ADP-glucose in vivo is considered to be
regulated by the [PGA]/[Pil ratio (Preiss 1982; Beck and Ziegler 1989).
Phosphorylation

of

the

smaller

subunit

of

spinach

ADP-glucose

pyrophosphorylase by a soybean calcium-dependent protein kinase has been

demonstrated in vitro (l'reiss et al. 1987), but in vivo phosphorylation has not, as
yet, been demonstrated. In maize leaves ADP-glucose pyrophosphorylase displays
a diurnal variation in activity which is not related to starch accumulation (Jeanette
arid Prioul 1992, 1994). These differences in activity were not related to changes
in the phosphorylation state of the enzyme, the quantity of the protein, or
differential responses of the mesophyll and bundle-sheath isoforms. Regulated
modifications in the specific activity of the enzyme were suggested to occur, but
the mechanism by which this was achieved remains to be elucidated.
Simultaneous synthesis and degradation of starch appear to occur in the light. In
addition, there appears to be a strong circadian influence in both starch and
sucrose accumulation. Li et al. (1992) found that starch accumulation ceased 2 h
before the end of light period while sucrose export increased at this time. These
authors suggested that the extra carbon exported as sucrose prevented carbon
accumulation as starch at this stage.
The relative rates of starch and sucrose synthesis are coordinated by the relative
distribution of the common inhibitor, Pi, and the two common precursors, triosc
phosphate and PGA, between the chloroplast and cytosolic compartments of the
cell. The flow of carbon appears to be preferentially directed to sucrose synthesis.
Carbon directed to sucrose synthesis competes for that directed to starch
accumulation, but the reverse does not occur (Stitt and Quick 1989). Thus, there is
a priority in carbon allocation in carbon partitioning between starch and sucrose
synthesis that favors the latter over the former. Starch accumulation can only
occur when the capacity for sucrose synthesis is saturated.

V. RELATIONSHIPS BETWEEN CARBON ASSIMILATION AND

CARBOHYDRATE SYNTHESIS IN SOURCE LEAVES
Photosynthesis, in air, is poised between limitation by ribulose-1,5-bisphosphate
carboxylase (RUBISCO) activity and limitation by the rate of ribulose-1,5bisphosphate

(RuBPI

regeneration. At

low

partial

pressures

of

CO,,

photosynthesis is limited by the carboxylation rate determined by RUBISCO

activity. At high CO, and saturating light, photosynthesis is limited by the capacity
of the BensonCalvin cycle to regenerate the substrate for carboxylation, RuBP.
The regeneration of RuBP is determined by the rate of electron transport that
provides the NADPH and ATP to drive the phosphoglycerate kinase, NADPglyceraldehyde 3-phosphate dehydrogenase, and phosphoribulokinase reactions.
In turn. ATP synthesis may be restricted by the availability of Pi. The pathways of
end-product biosynthesis utilize triose phosphate (TP) and regenerate Pi from
phosphorylated intermediates. Triose phosphate is the major end-product of
photosynthesis in the chloroplast. Under optimal conditions one-sixth of the TP is
exported or converted into starch within the stroma. The export of TP is strictly
regulated to prevent depletion of the levels of BensonCalvin cycle
intermediates that would severely impede the autocatalytic process. Transport
occurs across a specific translocator, the phosphate translocator, that exchanges
only divalent anions. It catalyzes the stoichiometric exchange of TP and some
other divalent anions, such as PGA, for Pi from the cytosol. One Pi molecule must
be returned to the stroma for every three molecules of CO, fixed. Triose phosphate
is converted to sucrose in the cytosol (Fig. 3), a process that liberates Pi. This is
returned to the chloroplast in exchange for more TP. In this way the processes of
photosynthesis and sucrose synthesis are coordinated (Fig. 3).
The Pi-translocator is restricted to photosynthetic tissues (Schulz et al. 1993).
Transgenic potato plants with decreased expression of the phosphate translocator
showed a 20%-30% reduction in Pi import capacity to the chloroplasts (Riesmeier
et al. 1993). The maximal rate of photosynthesis was decreased by 40%-60% in
these plants, and there was increased partitioning of assimilate into starch at the
expense of sucrose and amino acid synthesis (Riesineier et al. 1993).

From the preceding perspective feedback limitation of photosynthesis during

periods of limiting cytosolic sucrose synthesis would require that the stromal Pi
concentration falls sufficiently low as to restrict photophosphorylation and, hence,
CO, assimilation. It is virtually impossible to measure the free stromal Pi
concentration: perhaps the best approximation to the true in situ values for stromal
and cytosolic Pi were obtained by Sharkey and Vanderveer (1989) by using
nonaqueous fractionation of Phaseolus vulgaris leaves. They calculated the Pi
concentrations of these two compartments in Pi-deficient plants in air and under
feedback-limited conditions, that is, in leaves that exhibit 0 2-insensitive
photosynthesis. In air the stromal Pi concentration was approximately 7 mM, but
this value fell to about 2.7 mM when the leaves were held in low oxygen. Since 12 mM Pi in the stroma is bound to protein and, thus, not accessible to metabolism,
it is possible that the metabolically active Pi pool could fall low enough to limit
photophosphorylation. Interestingly, this study showed that the cytosolic Pi pool
was always greater than that in the chloroplast in the light.
The chain of events linking a decrease in sink demand to inhibition of CO 2
assimilation is only poorly characterized (Herold 1980; Geiger 1987; Foyer 1987;
Ho 1992). We must, therefore, consider the hypotheses that attempt to explain the
events leading to inhibition of carbon assimilation. Current hypotheses require the
following features:
1. That photosynthesis is responsive to the level of assimilate in source leaves
2. That low sink demand for assimilate generally causes accumulation of
assimilate in source leaves Interaction and Communication in Leaves 323
3. As a result key enzymes are either inhibited, inactivated, or decreased in
amount. This feedback limitation may involve
a. A decrease in chloroplastic Pi
b. A physical signal, possibly hydrostatic pressure or electrical signaling by
the phloem
c. Local or remote changes in apoplasticsympli.stic osmotic potentials
arid ionic status
d. Inhibition of phloem loading by increasing the sucrose concentration in
the veins or changes in hydrostatic pressure

e. Response of metabolism or gene expression in source leaves to growth

regulators
f. Response of metabolism or gene expression to sucrose or a derivative of
sucrose
4. These result in a decline in the rate of carbon assimilation. This may cause
decreases in the quantum yields of electron transport by photosystems I and II
0:1),,,/, (bpsid because of the empirical relationships that exist between the
latter parameters and the quantum yield of CO2 assimilation ()CO2).d
A. The Role of Inorganic Phosphate
The hypothesis that photosynthesis may be controlled by the level of assimilate in
source leaves is widely accepted, but the mechanism by which such regulation
may be achieved is debatable. The results of experiments in which attempts have
been made to block leaf export by heat-girdling, cold treatments, or leaf excision
must be viewed with extreme caution. Phloem transport is not blocked so
suddenly or completely in vivo, and such experiments represent very extreme
circumstances. In addition, factors such as the degree to which apoplastic and
symplastic pathways of export are inhibited may vary according to species, and
the capacity of a given plant species to overcome the girdling treatments is often
overlooked. however, such experiments have shown that the cessation of sucrose
export resuhs in an accumulation of sucrose in the leaf but does not cause an
increase in sucrose in the apoplastic compartment (Grusak et al. 1990; Ntiska and
Delrot 1986). It is generally supposed that under natural conditions low sink
demand for assimilate causes a decrease in assimilate export from source leaves
and hence an accumulation of assimilate. This, in turn, would decrease the rate of
photosynthesis (Harbinson 1994; Herold 1980; Pammenter et al. 1993). The
situation is, in reality, much more complicated than this simple scheme of events
may suppose, not least because plants often have several active sinks and can
store large amounts of carbohydrate as starch. However, it has long been supposed
that the Pi availability could be important in circumstances in which source
capacity exceeded sink demand (Herold 1980; Foyer 1987, 1988).

A Pi limitation in the chloroplast may be recognized by the following features:

1. Photosynthesis is no longer stimulated when the oxygen level of the
atmosphere is decreased from 21% to 2%; thus photosynthesis is 0 2insensitive.
2. Oscillations in photosynthetic flux become pronounced after transitions from
darkness to light or air to high CO,.
3. Phosphorylated intermediates accumulate in the leaves.
4. The situation may be reversed by supplying phosphate. However, note that
photosynthesis is inhibited when too much Pi is supplied through the petiole,
because of stomata! closure.
Pi-limitation arises in circumstances where the utilization of Pi exceeds the rate
of Pi-regeneration in end-product biosynthesis in the stroma and cytosol. The rate
of Pi-cycling is determined by the synthesis of end-products: sucrose in the
cytosol and starch in the chloroplast stroma (Figs. 3 and 4). Maximal rates of
photosynthesis cannot be maintained by Pi-cycling via stromal starch synthesis
alone, and the chloroplast is often considered to be more or less dependent on
cytosolic sucrose synthesis for its Pi supply. However, the capacity for
chhoroplastic Pi-cycling depends on the capacity for starch synthesis. The
partitioning of assimilate between starch and sucrose is dependent on the
interaction of three factors: (1) the concentration of the cytoplasmic Pi which
regulates the export of TP from the chloroplast, (2) the activity of the key
enzymes of the pathway of sucrose synthesis (FBPase, SPS, and possibly also
sucrose phosphate phosphatase), (3) the regulated activity of ADP-glucose pyrophosphorylase.
Ribulose-1,5-biphosphate carboxylase/oxygenase (RUBISCO) initiates both
photosynthetic carbon assimilation and the carbon oxidation cycle known as
photorespiration. In the latter phosphoglycollate is produced as a result of
oxygenation of RuBP. Conversion of phosphoglycollate to glycollate in the
chloroplast stroma liberates Pi. Phosphoglycollate formation and metabolism are,
thus, part of the Pi-cycling mechanisms in the mesophyll. Pi may also be an
important factor in the regulation of Rubisco activity in situ affecting the

carbamylation state of the enzyme. This has been suggested as a mechanism for
modulating photosynthesis via end-product accumulation (Sawada et al. 1992).
Leaf cells contain two distinct compartments of Pi, the metabolic pool in the
cytoplasm and the storage pool in the vacuole. Once sequestered in the vacuole,
the pool of Pi is not readily accessible to metabolism and turnover rates are low.
In darkness, the cytosolic Pi concentration is relatively large, but Pi is used for the
sugar phosphate syntheses during the initial minutes of photosynthesis and
subsequently photosynthesis becomes dependent on sucrose and starch synthesis
for Pi. The Pi-optimum for photosynthesis in isolated chloroplasts is extremely
sharp. Too much Pi will drain the BensonCalvin cycle of TP and inhibit the
regeneration of RuBP; too little Pi will cause a decrease in
photosynthesis because of Pi-limitation.
It is pertinent to examine the physiological circumstances in which the capacity
for carbon
assimilation exceeds that for sucrose and starch synthesis, and feedback limitation
of the overall rate of photosynthesis by Pi-availability occurs. There are two welldocumented situations when Pi-limitation of photosynthesis is manifest: low
temperatures and high atmospheric CO2 concentrations. Recent evidence suggests
that the phosphate status of the leaf is important in determining the temperature
response of photosynthetic carbon assimilation. When leaves are subjected to
suboptimal temperatures (lower than the growth temperature), a loss of the
characteristic stimulation of photosynthetic rate by 2% 0 2 is frequently observed
(Sharkey et al. 1986; Leegood and Furbank 1986). The lack of stimulation by 2%
02 is caused by a phosphate limitation of 'photosynthesis (Leegood and Furbank
1986; Labate and Leegood 1988). Feeding leaves with Pi enhances the
assimilation rate at low temperatures. High rates of photosynthetic CO,
assimilation at low temperatures require large pools of phosphorylated
photosynthetic intermediates (Leegood and Furbank 1986; Mchler et al. 1984;
Labate and Leegood 1988).

These observations are in accord with the observation that the Pi optimum for
photosynthesis in isolated intact chloroplasts increases at low temperatures
(Machler et al. 1984). Thus, the phosphate status of the leaf is modified by the
transition to suboptimal temperatures such that low temperature promotes much
higher levels of phosphate-esters and a decreased level of Pi. Seboptimal
temperatures, therefore, constitute one condition in which photosynthesis is
temporarily limited by the availability of Pi.
CO2 enrichment appears to produce symptoms similar to those observed in
Pi deficiency (Arp 1991; Betsche et al. 1989; Betsche et al. 1991; Yelle et al.
1989; Bowes 1991). CO2 enrichment initially greatly enhances the rate of CO,
assimilation. Generally starch synthesis increases dramatically even to levels that
are so high as to be deleterious to chloroplast structure and function (Stitt 1991).
In these circumstances assimilate utilization cannot keep pace with the CO 2
assimilation rate and this favors Pi limitation (Betsche et al. 1989; Morin et al.
1992; Duchein et al. 1993). High CO 2 also decreases the photorespiratory rate and
perturbs the sourcesink balance of the plant. Atmospheric CO2 enrichment
induces high levels of starch accumulation, low ATP/ADP and TP/PGA ratios, and
an increase in the photochemical quenching of chlorophyll a fluorescence
(Betsche et al. 1989). Increasing Pi availability to a level above the optimum
required in air considerably improves the growth response of a C 3 plant to
atmospheric CO2-enrichment.
Photosynthesis is limited by the capacity for sucrose synthesis in conditions
when light and CO2 are abundant. Indeed, photosynthetic rates can be improved
slightly by increasing the capacity for sucrose synthesis (Galtier et al. 1993,
1995). It is not clear, however, to what extent Pi limitation is responsible for the
inhibition of photosynthesis associated with accumulation of carbohydrate in the
leaf. 02 sensitivity is retained during inhibition of photosynthesis after source
sink manipulations in some species (Von Caemmerer and Farquhar 1984; Bagnall
et al. 1988). It is possible that accumulation of sucrose in the leaf causes
metabolite inhibition of sucrose phosphate phosphatase and SPS and this then
limits the recycling of Pi for continued photosynthesis. Feedback modulation of
photosynthesis in this way does not apparently occur to any great extent in

response to accumulation of carbohydrate. However, it is clear that carbohydrate

accumulation in leaves inhibits photosynthesis (Azchn-Bieto 1983; Plaut et al.
1987; Bagnall et al. 1988). In the short-term direct metabolic regulation of
component enzymes can lead to repression of the rate of CO 2 assimilation, but
recent evidence suggests that regulation is more likely to occur at the level of gene
expression (Krapp et al. 1991). in transgenic tobacco and Arabidopsis plants
where a yeast-derived invertase was expressed in the cell wall and in plants and
plant cell cultures fed with glucose, the coordinated reduction in the
photosynthetic apparatus preceded any functional feedback modulation or
photoinhibition. In these plants photosynthetic energy conversion and
photosynthetic capacity expressed on a chlorophyll basis remained high despite a
dramatic loss of chlorophyll (Von Schaewen et al. 1990; Krapp ei al. 1991;
Schiffer et al. 1992).
A chimeric yeast invertase gene has been expressed in the apoplastic
compartments of tobacco, Arebidnpsi,s tomato, and potato (Dickinson et al. 1991;
Sonnewald et al. 1991; Von Schaewen et al. 1990; I leineke et al. 1992). In all
cases phloem loading and export from source leaves were decreased, and this had
profound repercussions for plant growth and photosynthesis. The expression of
yeast invertase did riot affect photosynthetic metabolism in young leaves, while
the accumulation of starch and soluble sugars in the mature leaves was
accompanied by inhibition of photosynthesis related to the decrease in chlorophyll
and in the activities of enzymes involved in photosynthetic carbon metabolism
(Stitt et al. 1990; Von Schaewen et al. 1990). When yeast invertase was expressed
in the apoplast of potato leaves, the response of photosynthesis was rather
different (Heineke et al. 1992). Although photosynthesis was inhibited, this did
not appear to be due to either the feedback regulation of sucrose synthesis or
decreases in either chlorophyll or activities of enzymes or metabolite levels;
rather, it was related to the osmotic pressure in the leaf cells (Heineke et al. 1992).
B. Carbohydrate Accumulation
When an artificial limitation is imposed on the rate of sucrose export from
leaves, CO, assimilation frequently decreases (AzcOrr-Bieto 1983; Blechschmidt-

Schneider et al. 1989; Bagnall et al. 1988; Plaut et al. 1987; Sawada et al. 1986).
However, the relative effect depends very much on the species used and the
treatment performed. CO, assimilation may remain relatively unaffected (Van
Oene et al. 1992) or decrease to almost zero (Stitt et a1.1990). These differential
effects on CO2 assimilation may be correlated with differences in the use of
sucrose as the main storage carbohydrate (Haut et al. 1987; Goldschmidt and
Huber 1991). Where sucrose is the main storage carbohydrate the leaves appear to
show smaller decreases in the rate cf CO, fixation than leaves that accumulate
starch. The later are relatively more affected by decreases in sink capacity
(Goldschmidt and Huber 1992).
The carbohydrate status of the leaf changes diurnally and has been suggested
to have a circadian rhythm (Li et al. 1992; Weiner et al. 1992). It would appear
that elevated carbohydrate levels need to be sustained over long periods in order
to determine metabolic events under normal circumstances (Galtier et al. 1995).
Bearing this in mind, it is evident that there is a strong correlation between the
accumulation of carbohydrate and inhibition of CO2 assimilation (Azen-Bieto
1983; Sawada et al. 1986). The exact mechanism(s) whereby the sugar (sucrose,
glucose, or fructose) concentration is sensed and transduced is not known, and
much of the metabolite-mediated signal transduction process remains unresolved.
However, the discovery of metabolic repression of photosynthetic genes by
sucrose provided the first evidence at a molecular level for the intimate
association between the level of a photosynthetic end-product and the
transcriptional potential of the genes required for photosynthesis.
Metabolic regulation of gene expression is comparatively well understood in
Escherichia coli and yeast, but very little information is available on the
molecular mechanisms involved in higher plants. Sheen (1990) showed that the
transcriptional activity of seven photosynthetic gene promoters was specifically
and coordinately inhibited by the addition of sucrose, glucose, or acetate. Sugars
have been found to be positive or negative gene regulators in various plant tissues
(Koch et al. 1992). Sugar-mediated regulation is possibly a universal and
fundamental feature of the regulation of gene expression in higher plants. It is
interesting to note that glucose-mediated repression of the level of gene

expression is only about 10- to 20-fold in maize (Sheen 1990), possibly indicating
the need to regulate photosynthetic activity by this means, and not to eliminate it
altogether.
VI. EFFECTS OF SUCROSE PHOSPHATE SYNTHASE, NITRATE
REDUCTASE AND RESPIRATION ON BIOMASS
ACCUMULATION
It is widely accepted that sucrose phosphate synthase (SPS) has an important role
in the regulation of photosynthesis and assimilate partitioning in the leaf. Sucrose
phosphate synthase activity is inversely related to starch accumulation in leaves
and is correlated with assimilate export and plant growth. Changes in SPS activity
will have repercussions on photosynthesis, assimilate partitioning, export of
carbon from the leaf, and possibly growth and biomass production (Galtier et al.
1993; Foyer and Ferrario 1994; Foyer et al. 1995).
Transgenic tomato plants expressing both the native SPS and the SI'S gene from
maize were produced by Worrell et al. (1991) and Galtier et al. (1995). The active
enzyme was expressed from the cloned maize cDNA under the control of either
the promoter for the gene encoding the small subunit of RUBISCO from tobacco
(rbcs) or the promoter from cauliflower mosaic virus (CaMV promoter). In the
former, expression was largely limited to photosynthetic cells, while in the latter,
expression was constitutive. In the transgenic plants high levels of SPS were
always present. The diurnal modulation of the native tomato SPS resulted in a
marked decrease in SPS activity in the untransformed plants in the dark, but in the
transformed plants differences in enzyme activity between light and dark were
much smaller. In maize leaves SPS is known to be subject to lightdark
modulation regulated by protein phosphorylation. However, when maize SF'S is
expressed in tomato, this type of regulation is lost (Worrell et al. 1991; Galtier et
al. 1993). No detrimental effects of the increase in SPS activity were observed
(Galtier et al. 1993,1995). Carbon partitioning between starch and sucrose in the
leaves was modified in favor of sucrose formation (Fig. 6). Sucrose phosphate
synthase activity was found to be a major determinant of starch accumulation in
leaves as well as sucrose, and there was a strong positive correlation between the

ratio of sucrose to starch in the leaves and SPS activity (Fig. 6). Thus, SPS is a
major controlling factor determining assimilate partitioning in leaves. Carbon
partitioning between starch and sucrose was largely independent of irradiance, but
increases in the rate of sucrose synthesis were most pronounced at high irradiance.
Photosynthetic capacity was increased in plants expressing high SPS activity. The
increase in photosynthetic rate in air was not significant, but the light and
CO2saturated rate of photosynthesis was increased in leaves expressing high SPS
activity (Fig. 7). High SPS activity thus has the potential to increase ambient
photosynthesis when light and CO, are not limiting for CO, assimilation.
Estimated rates of carbon export from the leaf were remarkably similar in
transformed and untransformed plants irrespective of the growth irradiance or the
duration of the photoperiod. However, the transgenic lines expressing SPS under
the control of the CaMV promoter were distinct from those

Figure 6 The relationship between the leaf sucrose to starch ratio and SPS
activity in mature leaves of control and transgenic tomato plants. Plant
material was harvested from untransfomied controls () and high SPS
expressing transgenic tomatoes () during the light period. For other
details see Galtier et al. (1993). SPS, sucrose phosphate synthase.

Figure 7 The light response curves for photosynthesis in air (broken lines) and
in saturating CO, (solid lines) of leaves of high SPS-expressing transgenic
tomato plants (P, A) and untransfonned controls (0). See Galtier et al. (1993) for
details. SPS, sucrose phosphate synthase.
30

22
20
16
12
10

0
Control

Line 9

Line 13

Figure 8 Biomass production in untransformed wild-type controls and high

SPS-expressing transgenic tomato plants. In line 9 the maize SPS gene was
expressed under the control of the dies promoter; in line 13 expression of the
maize gene was constitutive. SPS, sucrose phosphate synthase.
expressing SPS under the control of the rbes promoter in terms of their relative
growth rate and biomass production (Fig. 8). Biomass production in the former

was greatly increased under all conditions, whereas it was similar to that of the
untransformed controls in plants expressing the SI'S gene in photosynthetic tissues
alone. This observation would suggest that there is a requirement for elevated SPS
in nonphotosynthetic tissues before overexpression of SPS can cause an increase
in biomass production. The increase in photosynthesis required in order to sustain
this increase in growth and biomass accumulation is only about 10% above that of
the wild type.
An increase in biomass is observed when the maize SPS gene is expressed
constitutively in all plant cell types (Fig. 8), but not when it is limited to
photosynthetic cells alone. Thus, although all types of high SI'S expressor show
elevated rates of sucrose synthesis and sucrose accumulation in leaves, this can
only he translated into increased biomass when SPS is also present at high levels
in nonphoto-synthetic cell types.
Biomass production, expressed as net carbon gain, is essentially governed by
the relative rates of photosynthesis and respiration. In many species a significant
portion (nearly half) of the carbon fixed during the photoperiod is used in dark
respiration. Indeed, there is a negative correlation between the rate of dark
respiration and net carbon gain. Inhibition of respiration by elevated CO, levels
may contribute to increases in net carbon gain in these circumstances. Biomass
accumulation is negatively correlated with leaf respiration and not with any other
obvious characteristic. This negative correlation between biomass accumulation
and dark respiration may result from uncoupled respiration that does not
contribute to plant growth. Respiration is not always tightly coupled to the
production of ATP and reducing equivalents. Part of the carbon is known to pass
through an alternative, cyanide-insensitive respiration pathway. This pathway is
found in all higher plants, although some plant tissues can express very high
levels of the alternative pathway and are thermogenic. Nonthermogenic plants are
in the majority, and the role of the alternative pathway in these plants is debatable.
Biomass production may, thus, be improved by decreasing respiratory losses. In
the case of the high SPS expressors respiratory rates were similar to those of the
untransformed controls. Biomass accumulation was, thus, favored by the increase
in carbon available for growth and development.

Biomass was not increased in transgenic Nicotiana plumbagenifolia with five

times higher levels of nitrate reductase (NR) activity. Neither did photosynthesis
appear to benefit from an excess of NR activity (Foyer et al. 1994a). The leaves of
the high NR expressors were not greatly different from those of the wild-type
controls with similar or very slightly increased rates of photosynthesis and
respiration,

comparable

SPS,

and

phosphoenolpynivate

carboxylase

(PEPcarboxylase) activities, and similar levels of protein, chlorophyll, starch, and

sucrose (Foyer et al. 1993, 1994). It is clear that the increases in NR activity were
insufficient to have much impact on photosynthesis, growth, or biomass
production. This suggests that in natural state NR is present in levels above those
required for maximal operation of the N assimilation pathway and production of
the photosynthetic components. Consequently, any further increase in NR activity
has only minor effects on the partitioning of carbon between amino acid biosynthesis and sucrose synthesis and is without significant effect on photosynthesis.
Our results suggest that overproduction of this single enzyme has only a marginal
effect on plant metabolism. In marked contrast, large changes in C and N
partitioning were observed in the plants expressing very low levels of NR activity,
where the ability to assimilate N clearly restricts growth and photosynthesis
(Foyer et al. 1994a).
VII. CARBONNITROGEN INTERACTIONS
Microalgae and higher plants assimilate nitrate by two closely linked steps:
(1) the uptake of nitrate from the environment into the cell and (2) intracellular
reduction of NO3 to NH,. catalyzed by nitrate reductase (NR) and nitrite
reductase. Ammonium is then incorporated into carbon skeletons via the
glutamine synthaseglutamine 2-oxoglutavate aminotransferase (GSCOGAT)
cycle. The mechanisms of NO, transport into cells, regulation of NR activity, and
coordination of carbon and nitrogen assimilation in photosynthetic cells are not
fully understood. The hypothesis that the pathways of carbon and nitrogen
assimilation compete for carbon skeletons and energy is widely accepted.
However, while there is evidence that this is the case in photosynthetic algae
(Hoppe arid "[lpin 1994), it appears that no such competition occurs in higher

plants, which are not exposed to the rapid changes in nutrient availability that
algae have to endure and exploit. Recent evidence suggests that continuous
reciprocal coregulation occurs between the pathways of carbon and nitrogen
assimilation in leaves (Huber et al. 1992b; Champigny and Foyer 1992;
MacKintosh and MacKintosh 1993). Precise regulation of these pathways at both
the genetic (Sugiharto and Sugiyama 1992; Sheen 1990) and metabolic levels
(Van Quy and Champigny 1992) allows concerted adaptive responses in the
pathways of carbon and nitrogen assimilation to changes in supply and demand.
The photosynthetic capacity of leaves correlates positively with their N
content, presumably because most N is used in the synthesis of components of the
photosynthetic apparatus. The relationships between photosynthetic carbon
assimilation and N assimilation in leaves have been studied extensively (Robinson
1988; Bloom et al. 1989; Khamis et al. 1990; Foyer et al. 1994b). In leaves
modulation of the activities of SPS, PEP-carboxylase, and NR has been shown to
contribute to the synchronization of N assimilation, CO, fixation, and carbon
partitioning (Kaiser and Spill 1991; Champigny and Foyer 1992; Huber et al.
1992a; Manh et al. 1993).
Nitrate is preferentially reduced in leaves, where it is the most important
source of N. Nitrate reductase is regulated in a complex manner by both shortterm modulation of activity and longer-term regulation of gene expression in
response to light and N supply. However, in almost all woody plants and in many
herbaceous plants at least part of total nitrate reduction occurs in the roots (Fig. 9).
The situation becomes even more complicated if microrhizal interactions are
considered. Our discussion of the CN interaction is limited largely to metabolic
events occurring in the leaves where photosynthesis drives N assimilation in the
light.

Light-regulated expression of the NR gene has been found to be mediated, at

least in part, by sucrose (Cheng et al. 1992; Vincentz et al. 1993), and metabolic
repression of transcription of seven maize photosynthetic gene promoters by
sucrose has been found (Sheen 1990). Sucrose is known to mimic the light
induction of NH gene transcription. In N. piumbaginifolio both N and C
metabolites affect the expression of the NH gene (Vincentz et al. 1993); Gln is an
inhibitor of NR gene expression, but exogenously supplied carbohydrates
antagonize this inhibitory effect. Glutamine (Gln) is also a positive metabolic
signal in the expression of PEP-carboxylase (Sugiharto and Sugiyama 1992;
Sugiharto et al. 1992). It not only affects PEP-carboxylase at the level of gene
expression but also causes changes in protein kinase activity (Manh et al. 1993)
and allosteric modulation (Schuller et al. 1990; Vanlerberghe et al. 1990).
Phosphorylation of SI'S and PEP-carboxylase appears to be a key regulatory
mechanism that diverts carbon flow to the anaplerotic pathway providing carbon
skeletons for N assimilation (Fig. 10). The phosphorylationdephosphorylation
changes are rapid and occur well in advance of the adaptive changes in protein
levels that enable the plant to support the increased demand for carbon residues.
Phosphoenolpyruvate-carboxylase activity is affected more than that of any other
enzyme by N availability. Whether N provokes a similar rapid response in NH
activation state remains to be elucidated.
Activation of cytosolic protein kinases has been proposed to divert
photosynthetic carbon from sucrose to amino acid biosynthesis (Fig. 10)
(Champigny and Foyer 1992). Sucrose phosphate synthase, PEPcarboxylase, and
NH have all been found to be regulated by protein phosphorylation (Van Quy et
al. 1991; Huber et al. 1991, 1992ab; Kaiser 1990; Kaiser et al. 1992). With regard
to SPS and PEPcarboxylase an increase in the level of phosphorylation has
inverse effects: SPS becomes inactivated while PEP-carboxylase is activated (Fig.
10). The partitioning of carbon between the pathways of carbohydrate and amino
acid synthesis thus involves inhibition of sucrose formation by inactivation of SPS
with concomitant activation of PEP-carboxylase (Van Quy et al. 1991; Van Quy
and Champigny 1992; Champigny and Foyer 1992). Glutamine (Gin) and
glutamate are the most likely metabolites controlling the activity of these enzymes

(Manh et al. 1993). These effectors have been shown to have positive and
negative effects, respectively, on the PEP-carboxylase protein kinase in vitro. The
hypothesis of Champigny and Foyer (1992) suggests that a derivative of nitrogen
assimilation in leaves, probably Gln, is a signal for the enhancement of protein
kinase activity.
Nitrate reductase has been shown to undergo rapid changes in activity with a
half-time of 10 to 20 min when leaves were subjected to lightdark transitions or
abrupt changes in CO, concentration (Kaiser and Brendle-Behnisch 1991; Kaiser
and Forster 1989; Riens and Heldt 1992). In transgenic Nicotiana plumbaginifolia
plants where there was fivefold increase in extractable NH activity because of the
constitutive expression of the NR gene, it was found that NH activity was
regulated in response to light in a similar manner to the native enzyme (Foyer et
al. 1994a; Vincentz et al. 1993). Nitrate reductase was ilia, tivated by
preineubation with ATP, but not with nonhydrolyzable ATP analogs. The
inhibition was only slowly reversed after ATP-removal, but recovery was greatly
accelerated by high concentrations of AMP or ethylettediamine tetraacetic acid
(EDTA); this suggested that the enzyme was inactivated by ATP-dependent
protein phosphorylation in the presence of Mg 2-i- and reactivated by
clephosphorylation of chelation of Mg 2+ (Kaiser and Spill 1991). The findings
were largely confirmed by Huber et al. (1992a), who were able to demonstrate
that the NH-protein is phosphorylated on seryl residues. Sonic of the auxiliary
proteins catalyzing these steps have been partially purified (Spill and Kaiser
1994), but it is still unclear how the activity of the protein kinases and
phosphatases is regulated. There is some evidence that NR-kinase is affected by
sugar phosphates (Hither et al. 1993), much as SPS-kinase is (Weiner et al. 1992).
Dephosphorylation in vivo was completely prevented by pretreating leaves with
okadaic acid (Huber et al. 1992), as was activation of NH in pea roots (Glaab and
Kaiser 1993), indicating involvement of a type 1 or 2A protein phosphatase. Only
protein phosphatase (PP) 2A can dephosphotylate

and activate Nit (MacKintosh 1992), suggesting a specific role for PP 2A and not
PP 1. Mg2+ appears to play an important role in NR regulation. The NR protein
phosphatases are progressively inhibited by free Mg 2+ above 0.5 mM, and the
ATP-dependent NR inactivation by the protein kinase depends on free Mg 2+
(Kaiser and Spill 1991; Spill and Kaiser 1993).
In animal cells, the central enzyme of a protein kinase cascade is a 5-AMPstimulated protein kinase (MacKintosh and MacKintosh 1993). In plants 5-AMP
has been shown to accelerate NR reactivation (Kaiser and Spill 1991; Kaiser et al.
1992; Glaab and Kaiser 1993); therefore, AMP may activate the NR protein
phosphatase. Reactivation was stimulated by chelation of divalent cations (Kaiser
and Spill 1991; Huber et al. 1992).
Nitrate reductase activity is decreased in maize roots when aerial parts of the
plant are deprived of CO2. This can be traced to a limitation on the carbon supply
to the roots (Pace et al. 1990). The necessity to control NR in roots rapidly is less
obvious than in leaves, as fluctuations in the carbohydrate supply to roots in
response to environmental conditions will probably be slower than in leaves.
However, the enzyme in roots has been shown to be modulated in response to
oxygen supply, with a half-time as short as in leaves. Rapid modulation of root
NR is probably achieved by a similar mechanism to that occurring in leaves
(Clash and Kaiser 1993).
VIII. CONCLUSIONS AND PERSPECTIVES
Considerable progress has been made in recent years toward understanding the
processes involved in sourcesink interactions in higher plants at the
biochemical, physiological, and molecular levels. Much of this information has
been obtained through manipulation of single enzyme components in transgenic
plants in order to answer questions relating to the nature and significance of
putative control points. At present sourcesink relationships are modified as a
result of cultural practices such as removal of fruit to increase the size of
remaining fruit, spraying with gibberellic acid to reduce the number of flowers
that set, and trunk girdling to enhance fruit maturity. Such alterations of the plant's
natural sourcesink relationships commonly occur in the production of certain

crops, particularly perennial horticultural plants. However, in order to engineer

plants specifically to improve performance and yield we need a much better
understanding of the interacting biochemical and physiological processes in this
very complex system.
Accumulation of carbohydrate in leaves is potentially a lethal situation, but it
appears to be intimately involved with the adaptive changes in enzyme activities
that enable the plant to support changes in demand for assimilated carbon. Rapid
transitory metabolic responses to changes in demand occur, but they do not
normally appear to be associated with carbohydrate accumulation in the leaf.
While the theory of feedback control requiring decreases in Pi availability (Herold
1980; Foyer 1987) is attractive, Pi limitation does not appear to result from
carbohydrate accumulation. It is easy to appreciate why this may be the case since
severe Pi limitation compromises photosynthetic function and potentially
predisposes the plant to photoinhibition and photooxidation (Foyer et al. 1990). A
much more logical solution would be to reallocate the carbon accumulating in the
leaf, or, if that proves impossible, to regulate the capacity for carbon assimilation
by reducing the amount of photosynthetic machinery' in operation. This is not to
say that Pi is not of central importance to the coordination of carbon assimilation,
carbon partitioning, and electron transport capacity. It is evident from classical
biochemical studies and from work with transgenic plants that by increasing the
rate of TP utilization (and, hence, the rate of Pi-recycling) increases in the rate of
photosynthesis occur when light and CO, are not limiting for this process. Thus,
there is potential for increasing photosynthesis and increasing biomass production
by manipulation of the rate of Pi recycling via increased sucrose synthesis (Galtier
1993, 1995). While Pi is an important component of sourcesink coordination
and assimilate partitioning at the intracellular level, it does appear to be involved
in the decrease of photosynthesis resulting from carbohydrate accumulation in the
leaf.
Variations in demand for assimilate elicit changes in the steady-state levels of
component proteins. This may be achieved in at least two ways: (1) by
accumulation of soluble carbohydrate that acts at the level of transcription and (2)
by mediation of hormonal factors such as gibberellins. This is evidence for a role

for mediation of both kinds. In this review we have limited our discussion to
consider the former type of regulation. However, gibberellic acid has been shown
to regulate the steady-state level of SPS and, hence, its activity in both soybean
and spinach leaves (Cheikh et al. 1992). Hormonal control of protein synthesis
may, thus, be an intrinsic feature of the sourcesink interaction that coincides
with and amplifies the effects of carbohydrate accumulation (Roper and Williams
1989). Abscisic acid is well known as a regulator of strornal aperture, but it also
may affect other processes within the leaf associated with CO, assimilation in an
indirect manner.
Multiple step interactions among hormones, sugars, amino acids, and light
modulate the expression of photosynthetic genes and those involved in nitrogen
assimilation. The transcription of NR messenger ribonucleic acid (mRNA) in
Arabidopsis and tobacco requires high light. Light can be replaced by exogenous
sugars, although the effects of low light suggest that there is also a phytochromemediated component. Cytokinin enhances the steady-state levels of specific
photosynthetic proteins and also enhances the accumulation of NR mRNA (Suty
et al. 1993).
The rather slow, adaptive down-scaling of photosynthetic capacity following a
decrease in sink demand clearly confers a physiological advantage. It prevents
deleterious effects that might otherwise occur if the thylakoid membranes were
suddenly deprived of substrates. It allows optimization of resources while
conferring a degree of flexibility that is essential if the plant is to take maximum
advantage of its autotrophic state. Many factors act together to provide precise
coordination of source and sink. While the sugar concentration is one metabolic
trigger that has been demonstrated to modify metabolism and affect gene
expression, the content of sucrose or glucose alone is not a decisive factor (Galtier
et al. 1995); rather the ratio of sugars to amino acids such as Gln in the
perspective of the prevailing hormonal balance is. This view in itself may he nave
since signals of other types will undoubtedly affect the influence of these intrinsic
metabolic regulators.

ABBREVIATIONS
(1)co_relative quantum efficiency of CO, assimilation
CIS tindex of the relative quantum efficiency for electron transport by PS I
4

"Ps tiindex of the relative quantum efficiency for electron transport by PS II

DHAI)dihydroxyacetone phosphate
E41)

erythrose 4-phosphate

Ed

fcrredoxin

E6P

fructose 6-phosphate

FBP fructose 1-6 bisphosphate

FBPasefructose 1-6 bisphosphatase
GAP glyceraldehyde phosphate
GBP glycerate-1, 3 bisphosphate
GOGATglutamine 2-oxoglutarate amino transferase
Gln

glutamine

GS

glutamine synthase

GIP

glucose 1-phosphate

G61'

glucose 6-phosphate
product of (Dps I and incident irradiance

is IIproduct of (Lips and incident irradiance

N

nitrogen
4.

Interaction and Communication in Leaves

NADP-MDII NA DP-malate dehydrogenase
NR

nitrate reductase

P700fraction of the P.), pool that remains unoxidized

P7004

fraction of thepool that is oxidized

PEP phosphoenolpyruvate
Pi

inorganic phosphate

PGA 3-phosphoglycerate
PP

protein phosphatase

PQ

plastoquinone

PS I

photosystern I

335

PS II photosystern II
primary stable electron acceptor of PS II
q(2coefficient for photochemical quenching of chlorophyll a fluorescence
qNPcoefficient for nonphotochemical quenching of chlorophyll a fluorescence
RSP ribose 5-phosphate
IN BPribulose-1,5-bisphosphate
RUBISCOribulose-1,5-bisphosphate carboxylase/oxygenase
S7Psedoheptulose 7-phosphate
S13Psedoheptulose-1,7-bisphosphate
SI3Pasesedohepulose-1,7-bisphosphatase
SI'Ssucrose phosphate synthase
SS

sucrose synthase

TP

triose phosphate

Xu5Pxylulose 5-phosphate

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