Download Sucrose metabolism: regulatory mechanisms and pivotal roles in

Sucrose metabolism: regulatory mechanisms and pivotal roles
in sugar sensing and plant development
Karen Koch
Sucrose cleavage is vital to multicellular plants, not only for the
allocation of crucial carbon resources but also for the initiation of
hexose-based sugar signals in importing structures. Only the
invertase and reversible sucrose synthase reactions catalyze
known paths of sucrose breakdown in vivo. The regulation of
these reactions and its consequences has therefore become
a central issue in plant carbon metabolism. Primary mechanisms
for this regulation involve the capacity of invertases to alter
sugar signals by producing glucose rather than UDPglucose,
and thus also two-fold more hexoses than are produced by
sucrose synthase. In addition, vacuolar sites of cleavage
by invertases could allow temporal control via
compartmentalization. In addition, members of the gene families
encoding either invertases or sucrose synthases respond at
transcriptional and posttranscriptional levels to diverse
environmental signals, including endogenous changes that
reflect their own action (e.g. hexoses and hexose-responsive
hormone systems such as abscisic acid [ABA] signaling). At the
enzyme level, sucrose synthases can be regulated by rapid
changes in sub-cellular localization, phosphorylation, and
carefully modulated protein turnover. In addition to
transcriptional control, invertase action can also be regulated
at the enzyme level by highly localized inhibitor proteins and
by a system that has the potential to initiate and terminate
invertase activity in vacuoles. The extent, path, and site of
sucrose metabolism are thus highly responsive to both
internal and external environmental signals and can, in turn,
dramatically alter development and stress acclimation.
UDP
UDPG
VIN
VPEc
Addresses
Plant Molecular and Cellular Biology Program, Horticultural Sciences
Department, University of Florida, Gainesville, Florida 32611, USA
e-mail: [email protected]
Figure 1
uridine di-phosphate
UDPglucose
vacuolar invertase
vacuolar processing enzyme g
Introduction
The only known enzymatic paths of sucrose cleavage in
plants are catalyzed by invertases (sucrose þ H2O !
glucose þ fructose) and sucrose synthases (sucrose þ
UDP ! fructose þ UDPglucose) (Figure 1). Both of
these paths typically degrade sucrose in vivo but the
products of their reactions differ in important ways
[1,2]. Invertases produce glucose instead of UDPglucose
(UDPG), and thus also form twice as many hexoses.
Either of these features could give invertases a greater
capacity to stimulate specific sugar sensors [3–9]. The
resulting signals can alter expression of diverse genes
[3,10,11], so invertases can potentially be strong effectors
of widely varying processes. These include the biosynthesis and perception of hormones such as abscisic acid
(ABA). In addition, both sugar and hormone signals can
also affect the expression of the genes that encode sucrose
synthase and invertase [1–3,5,7,12,13,14,15,16,17]. This
review explores the hypothesis that sucrose metabolism
lies at the heart of a sensitive, self-regulatory developmental system in plants. Its influence appears to be
balanced by the capacity for sensing sucrose itself,
Hexose signals
Glucose
Current Opinion in Plant Biology 2004, 7:235–246
This review comes from a themed issue on
Physiology and metabolism
Edited by Christoph Benning and Mark Stitt
1369-5266/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
Invertase
Fructose
Hexose
signals
Sucrose
Sucrose synthase
Fructose
UDP glucose
?
Sucrose signals
Current Opinion in Plant Biology
DOI 10.1016/j.pbi.2004.03.014
Abbreviations
ABA
abscisic acid
CIN
cytoplasmic invertase
CWIN cell wall invertase
ENOD early nodulation
PPi
inorganic pyrophosphate
PPV
precursor protease vesicle
SnRK SNF-related kinase
SUS
sucrose synthase
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The sucrose-cleaving enzymes are pivotal to maintaining the balance
between both sugar signals and metabolic paths. Different sensing
mechanisms are activated by hexoses and by other downstream
metabolites, and also by sucrose itself. The invertase path of
sucrose cleavage generates free glucose and twice as much overall
substrate for hexose-based sugar sensing as does the degradative
action of the reversible sucrose synthase reaction (i.e. two hexoses
as opposed to fructose þ UDPG). In addition, genes that encode
sucrose-cleaving enzymes are responsive to the action of their own
enzyme products. They respond to sugar signals in addition to
affecting the generation of these signals.
Current Opinion in Plant Biology 2004, 7:235–246
236 Physiology and metabolism
but different systems and responses are involved
[18,19,20,21,22].
Additional factors in the link between sucrose metabolism and sugar signals lie in the physical path of sucrose
import and sites of sucrose cleavage (Figure 2). Sucrose
can move from phloem into the cytoplasm of sink cells
with or without crossing the plasmamembrane or the cell
wall space. This is an important distinction because
points of membrane interface have been implicated in
specific mechanisms of both sucrose and hexose sensing
[1,4,8,22,23]. The plasma membrane can be exposed to
abundant sucrose and/or hexoses if plasmodesmatal connections between cells are absent [3,4] (or functionally
limited [24]). Cell wall invertase (CWIN) can markedly
increase localized hexose levels in these instances, which
typify developing seeds and grains [6,25,26] in which
there is no symplastic (plasmodesmatal) continuity
between maternal and seed tissues. Pathogens and other
specific stimuli can also induce cell wall invertases, even
when plasmodesmatal paths are intact [5,17,27], and
Figure 2
Phloem
Sucrose
Cell wall
space
Sink cell cytoplasm
Vacuole
Plasmodesmata
Sucrose
these instances can favor sucrose import and amplification
of hexose signals if sucrose moves out into the extracellular space.
In contrast, plasmodesmatal continuity remains intact
and provides the predominant transfer path for sucrose
throughout most maternal sinks (i.e. roots, shoots, fruit
flesh and so on) ([3–5]; Figure 2). In these tissues, sucrose
can be imported with minimal effect on known signaling
mechanisms [3,4]. Once inside the importing cells,
sucrose metabolism again becomes important to sugar
sensing, this time to endogenous hexose- and possibly
sucrose-signaling systems [4,5,10,11,22,28]. The products of sucrose synthase (SUS) action initiate the fewest
hexose-based signals [3,6] (which is advantageous under
many conditions [8,17,29–31]), and cytoplasmic invertases (CINs) are minimally active in most systems [2].
However, cytoplasmic sucrose is frequently transported
into vacuoles for cleavage.
Vacuolar invertases (VINs), like their cell wall counterparts, generate abundant hexoses and hexose-based sugar
signals [5,9,27,32]. The VINs also mediate the primary
path of sucrose cleavage in expanding tissues [1,2], and
thus contribute to considerable hexose flux across the
tonoplast and to the entry of hexoses into cytoplasmic
metabolism. Temporal control of both processes is further
facilitated by vacuolar compartmentalization, which
could integrate the import and signaling functions of
VIN with its osmotic role in expansion.
Sucrose-cleaving enzymes can alter plant
development through sugar signals
CWIN
CIN
G+F
G+F
SUS
UDPG + F
VIN
G+F
Current Opinion in Plant Biology
The physical path of sucrose movement and site of its cleavage are
central not only to mechanisms of import but also to the sugar signals
generated. The mechanisms that regulate sucrose movement and
cleavage also differ for each compartment. Depending on the path of
sucrose entry into an importing structure (i.e. via plasmodesmata or
across the cell wall space), sucrose can be cleaved by cell wall
invertase (CWIN), cytoplasmic invertase (CIN), sucrose synthase (SUS),
or vacuolar invertase (VIN). Cytoplasmic sucrose cleavage typically
produces limited hexoses or hexose-based sugar signals because
of the generally low activity of CIN and the production of UDPG instead
of glucose by SUS. In contrast, sucrose that enters sinks via the cell
wall space can generate significant amounts of glucose and other
hexoses if CWIN is active. Glucose and fructose can, in turn, initiate
hexose-based signals both at the membrane and during subsequent
metabolism in the cytoplasm. Similarly, abundant hexoses and
hexose-based signals can be generated in the vacuole by VIN; this
constitutes the primary site of sucrose cleavage during the expansion
phase of most sink tissues. F, fructose; G, glucose.
Current Opinion in Plant Biology 2004, 7:235–246
In addition to their production of metabolic substrates,
each site and path of sucrose cleavage described above
can initiate a distinctive profile of sugar signals, which
in turn can have profound developmental effects
(Figure 3). In general, hexoses favor cell division and
expansion, whereas sucrose favors differentiation and
maturation [6,26,33,34]. These effects, together with
information from analyses of numerous systems, has led
to an invertase/sucrose-synthase control hypothesis for
transitions through the prominent stages of development [6,26,33,34]. According to this hypothesis, invertases mediate the initiation and expansion of many new
sink structures [5,9,35], often with vacuolar activity preceding that in cell walls [17,36]. The action of cell wall
invertase coincides with the elevated expression of hexose transporters in some systems [23]. Later transition to
storage and maturation phases is facilitated by changes in
the hexose/sucrose ratio (i.e. the cellular ‘sugar state’
[6,26,33,34]), and by shifts from invertase to sucrosesynthase paths of sucrose cleavage. In some highly localized regions, elevated levels of cell wall invertase persist
during maturation [17,37]. Sucrose synthase, too, can be
active in localized sites during the early phases of development (e.g. in the phloem and at sites of rapid cell wall
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Sucrose metabolism Koch 237
deposition [17,38–40]). Overall, however, diverse evidence supports the contention that the balance between
invertase and sucrose synthase activity can alter plant
development through differential effects on sugar signaling systems.
Relative contribution
to sucrose metabolism
Figure 3
Vacuolar
invertases
Cell wall
invertases
Sucrose
synthases
Developmental progression
Vacuolar invertases:
Sink initiation and expansion
Maximal generation of vacuolar hexoses
- Expansion (linked to photosynthate availability)
- Respiration
- Hexose-based sugar sensing
(cell division and initial differentiation)
Cell wall invertases:
Continued sink initiation and expansion
Maximal generation of cell wall hexoses
- Reduction of turgor-based transport inhibition,
especially as volume increases to near maximum
- Turgor reduction in zones of phloem unloading
(localized action often in much of development)
- Respiration
- Hexose-based sugar sensing
(cell division and related gene expression)
Invertase-derived hexose signals can also markedly alter
the expression of genes for both the biosynthesis and
sensing of specific hormones (e.g. ABA). Genes for both
processes can be regulated at transcriptional and posttranscriptional levels by hexose signals [11,41,42]. In
some instances, effects on one hormone system may
indirectly influence others (e.g. antagonisms between
ABA and ethylene or auxin [11,41–43]). Nonetheless,
the influence of sugar-sensing systems extends from
cytokinins [11,42] to gibberellins [42,44], and from auxin
[11] to ethylene [10,41–43,45]. Some thorough and striking studies of the sugar–ABA interface have been carried
out recently. Here, hexose-based signals (originating from
sucrose cleavage) are implicated in regulation of ABA
biosynthetic genes [45], in the early steps of ABA sensing
[46], and in influencing downstream elements in this
signal transduction system [46,47].
The influence of sucrose cleavage products on hormone
systems combined with the regulation of sucrose metabolism by those hormone systems together comprise a
means of integrating individual cellular responses into
those of the multicellular organism.
Sucrose synthases:
Storage and maturation
Generation of UDPG instead of glucose
- Direct shuttling of UDPG to biosynthesis
- ATP-conserving respiratory path
- Minimization of hexose-based sugar sensing
(enhanced expression of genes for
storage and maturation)
Current Opinion in Plant Biology
A common developmental profile of the contributions of sucrosecleaving enzymes to sequential stages of sink initiation, expansion, and
storage/maturation. Both vacuolar and cell wall invertases maximize
hexose production, which in turn enhances respiration and the
generation of hexose-based signals. These hexose-based signals
upregulate diverse early development genes, including those
involved in cell division. Vacuolar invertases concurrently favor cell
expansion through their two-for-one enhancement of osmotic
solutes in the vacuoles, as well as by linking the initial volume
increase to photosynthate availability. As cell expansion slows, the role
of cell wall invertase becomes increasingly important in preventing the
turgor-based inhibition of symplastic transfer, and also in reducing
turgor in the zones of phloem unloading. As the cell division and
expansion phases shift to those of storage and maturation, sucrose
synthases become more important, and contribute in at least three
overall areas. These are their capacity to direct carbon to an ATPconserving respiratory path that is advantageous in many sink
tissues, to facilitate the shuttling of UDPG to biosynthetic products
or other special functions, and to minimize the production of hexoses
and thus hexose-based signals during specific stages of sink
development.
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Roles and regulation of sucrose synthases
Essential roles of sucrose synthases
Recent developments indicate that the role of sucrose
synthase in sucrose import may involve a dual capacity to
direct carbon toward both polysaccharide biosynthesis
and an adenylate-conserving path of respiration. A key
function of sucrose synthase in biosynthetic processes is
supported by evidence of its contribution to cell wall
formation, which is inhibited in maize sucrose synthase
knock-outs (K Koch et al., unpublished), maize mutants
[2,48], anti-sense carrot plants [5], and anti-sense cotton
seeds [40]. The work on cotton [40] showed that highcellulose fibers do not form if sucrose synthase activity has
been significantly decreased; and where antisense effects
extend from epidermis to the seed interior, embryo and
endosperm development are also inhibited. Additional
analysis of mutant and antisense plants that had reductions in sucrose synthase (compiled in [9]) also showed
that these plants had markedly less starch in storage
organs (e.g. carrot roots and potato tubers). The UDPG
product of sucrose synthase has been implicated in the
formation of the starch [49] and in the synthesis of callose
[39,50] and diverse cell wall polysaccharides [51,52].
The centrality of sucrose synthase to sucrose import by
many structures may relate to the typically moderate, but
widespread, reductions of oxygen levels within certain
Current Opinion in Plant Biology 2004, 7:235–246
238 Physiology and metabolism
structures. Recent data indicate that endogenous oxygen
levels are generally reduced to varying degrees in active
sinks such as potato tubers [53] and developing seeds
[54], and also in the phloem [55]. Sucrose synthase can
operate effectively under these conditions [7], and can
even reduce the extent of oxygen depletion [53], but
invertases typically do not operate well under these
conditions [7]. The activity of invertases can also exacerbate the problem of oxygen depletion [53]. Evidence
from transgenic potatoes shows that the normal developmental shift to sucrose cleavage by sucrose synthase in
wildtype tubers improves not only their adenylate balance, starch biosynthesis, and respiratory costs, but also
their endogenous oxygen levels relative to those of tubers
that overexpress alternate sucrose breakdown enzymes
(i.e. invertase or sucrose phosphorylase) [53]. Sucrose
synthase is one of only a few genes to be upregulated
under low-oxygen conditions [8,30]; its essentiality under
these conditions is indicated by the capacity of wildtype
plants but not sucrose-synthase-deficient mutants to survive flooding [56,57]. Under pronounced low-oxygen
conditions, sucrose synthase responds rapidly to early
rises in cytosolic Ca2þ [31,50] and can support considerable biosynthesis (of cellulose and callose) [50,51]. The
functional significance of sucrose synthase, as opposed to
invertase, is likely to be particularly important in lowoxygen conditions where it can aid the respiratory conservation of adenylates through the production of UDPG
instead of additional hexoses (which would each require
an ATP for entry into glycolysis).
Sucrose synthases may also be central to the efficacy of
at least some symbioses (e.g. nitrogen-fixing nodules
[58,59]) and have been implicated in the maintenance
of others (e.g. mycorrhizae). A rug4-a (rugosus meaning
wrinkled) mutation that reduces sucrose synthase levels
in pea seeds and nodules inhibits nitrogen-fixation [58];
conversely, if soybean nodules harbor symbionts that are
unable to fix nitrogen, then sucrose synthase is not
induced [59]. Sucrose synthase and VIN are also induced
specifically in root cells that have mycorrhizal arbuscules
[60] (and sometimes also in adjacent cells [61]), and this
upregulation occurs early in development [62].
Further roles for sucrose synthase in development and
sucrose import by diverse sinks have been implicated.
The sucrose synthase gene is one of the first to show
elevated expression as leaf primordia differentiate from
the apical meristem [63]. The leaves and roots of carrot
plants in which sucrose synthase has been reduced transgenically are also ultimately smaller than those of wildtype plants [5], and similar tomato transgenics showed
reduced fruit set from the earliest flowers [64]. A sucrose
synthase path of sucrose cleavage also typically predominates over those involving invertases during the storage
and maturation stages of organ development [65,66]. At
least some of these developmental effects are likely to
Current Opinion in Plant Biology 2004, 7:235–246
stem from the capacity of sucrose synthase to minimize
hexose-based sugar signals, particularly during periods
when these signals could be detrimental to differentiation
and/or maturation [6].
It is also worth noting that sucrose synthase may be
significant to human nutrition in a context not previously
appreciated: the amino acid content of this protein,
together with its abundance in mature grains, make it
an important contributor to nutritionally limiting lysine
levels in maize kernels [67]. Approximately 75% of total
kernel lysine is contributed by sucrose synthase and two
other cytoskeleton proteins (UDPG starch glucosyl transferase and fructose 1,6-bisphosphate aldolase).
Sucrose synthase’s role in phloem sieve elements
Recent work has immunolocalized sucrose synthase to
sieve-tube elements as well as to companion cells [17],
thus linking sucrose cleavage to sieve-tube function
more directly than previously recognized (Figure 4).
Earlier studies had localized sucrose synthase to adjacent
companion cells [68], a site compatible with the results of
metabolite analysis that indicated activity in or near the
transport path [69]. Later work also showed that inorganic pyrophosphate (PPi) is essential for phloem function, and implicated a sucrose-synthase-based path of
respiration, probably in companion cells [70]. However,
recent results move sucrose synthase into close physical
proximity with the sieve-tube plasma membrane and a
phloem-specific ATPase that is believed to aid sucrose
transport and compartmentalization [17]. This sievetube locale could also facilitate sucrose synthase’s probable role in directly supplying UDPG for the rapid
wound-induced biosynthesis of callose plugs. Further
advantages of sucrose synthase in both sucrose transport
and callose biosynthesis are its capacities to favor the
cycling of PPi as opposed to ATP [29,55,69] and to
function effectively under low oxygen conditions
[30,56]. Both PPi cycling and low oxygen conditions
are typical of the phloem [55,69]. The actin and/or
membrane association of sucrose synthase [2,71,72,73]
may also provide an important physical anchor for its
action within the phloem transport stream.
Transcriptional control of sucrose synthases
Several advances have arisen from attempts to unravel the
basis of the differential expression of sucrose synthase
genes, particularly in response to oxygen and sugar availability. Those sucrose synthase genes that are upregulated by carbohydrate deprivation are also the most
strongly induced by low-oxygen, and show narrow patterns of expression that could confer sucrose import or
survival priority for key tissues [3,8,30]. The starvationinducible maize sucrose synthase (Shrunken1 [Sh1]) also
respond rapidly to low O2, with marked increases in both
its mRNA levels and enzyme activity, especially under
modest oxygen depletion (i.e. 3% O2) [30]. Additional
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Sucrose metabolism Koch 239
Figure 4
work on sugar-inducible sucrose synthases in potato has
indicated that this upregulation requires SNF-related
kinase (SnRK), a SNF1 ortholog, because the response
was abolished at the mRNA level in SnRK-antisense
plants [74–76]. Although data from analogs that cannot
be metabolized suggest little or no hexokinase involvement in the induction of rice Rsus1 [77], separate roles for
both hexokinase-dependent and -independent sugarsensing mechanisms are implicated in the responses of
an Arabidopsis Sus1 [78].
(a)
Sucrose synthase
Sieve-tube element
Open
lumen
Companion cell
(b)
Cell
wall
Sieve-tube cytoplasm
Sievetube
lumen
Sucrose
UDP
Sucrose synthase
Biosynthesis
Respiration
UDPG Fructose
PPi
Callose
synthase
Translational control of sucrose synthase
UGPase
A key contributor to sustaining elevated levels of sucrose
synthase mRNA and protein under low oxygen appears to
be the preferential loading of sucrose synthase mRNAs
onto large polysomes [80], where their translation is enhanced by upregulation of both initiation and elongation
[80]. Sucrose synthase activity increases rapidly but to
varying degrees under low oxygen [30], but a still-greater
capacity for activity increase after stress release is indicated
by the high levels of accumulated mRNA. Such disparities
between regulation at transcriptional and posttranscriptional levels may have specific physiological significance.
PPi
F6P
UTP
G1P
?
Glycolysis
?
Callose
for plugging
One of the strongest known plant enhancers of gene
expression is the first intron of the Sh1 maize sucrose
synthase, which is particularly effective when introduced
into the 50 region of a gene or construct. Recently, a 145bp portion of the 1028 bp Sh1 intron was found to induce
gene expression by 20–50-fold. Most of this induction was
mediated by the T-richness of a 35-bp motif rather than
its specific sequence [79].
ATP
for transporter
ATP
ADP
Sucrose
H+
H+
Current Opinion in Plant Biology
Sucrose metabolism in phloem sieve-tube elements. (a) Sucrose
synthase localization extends to the sieve-tube element, where it
visually co-localizes with phloem-specific membrane ATPases [17]
and lies in close proximity to sucrose transporters and sites of rapid
wound-induced callose formation. The sieve-tube lumen is open to
solute flow, but detectable levels of sucrose synthase do not move
within it [69]. The capacity of sucrose synthase to bind actin [72] and
membranes [2,52,73,84,85,90], and/or to localize in specific regions
of the cytoplasm [39] is compatible with its minimal mobility and highly
localized presence on or near the sieve-tube plasmamembrane [17].
(b) A model for the regulation of sucrose use within sieve tubes
integrates recent information on the cellular and sub-cellular localization
of this sugar with the results of analyses of metabolic changes in the
phloem sap [66,70] and internal environment [55]. Two of the greatest
demands for sucrose use are likely to be callose biosynthesis, which is
massive during defensive plugging, and ATP production for the
maintenance of steep sucrose gradients across the plasma
membrane. Both processes could be readily supported by products of
the sucrose synthase reaction with minimal input from local
mitochondria, especially if PPi levels favor the activity of UDPG
pyrophosphorylase (UGPase). The resulting uridine tri-phosphate
(UTP) can contribute either directly or indirectly to glycolysis and ATP
formation. The alternative demands made by callose biosynthesis
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Regulation of sucrose synthase by sub-cellular
localization
Recent developments in determining how the membrane
localization of sucrose synthase is controlled are presaged
by earlier work on the enzyme’s apparent association with
rosettes of the plasma membrane cellulose synthase complex (Figure 5). Such a relationship could allow UDPG
from sucrose synthase to feed directly into cellulose formation, and essential UDP to be recycled readily [81].
Evidence indicates that the involvement of sucrose
synthase in this rapidly changing structure [38,52] might
also extend to the analogous formation of mixed-linkage
poly-glycans [82], and to the production of callose near the
phragmoplast or in localized ‘exoplasmic zones’ [39,52,83].
Recent work indicates a still-broader role for the transient
association of sucrose synthase with membranes, and
further, that the control of this localization might
in sieve tubes could be closely associated with sucrose synthase
through both PPi cycling (from callose synthase back to UGPase)
and with the proximal localization of sucrose synthase, UGPase, and
callose synthase [39]. F6P, fructose-6-phosphate; G1P, glucose-1phosphate.
Current Opinion in Plant Biology 2004, 7:235–246
240 Physiology and metabolism
Figure 5
Plasma
membrane
(a) Cell wall
Cytoplasm
Cellulo
se
Sucrose
Sucrose synthase
Cellulose synthase complex
(b) Cell wall
Plasma membrane
ose
llul
Ce
Pore
subunit
Cytoplasm
UDPG
UDPG
UDP
UDP
Cellulose
synthase
Fructose
Sucrose
Amyloplasts
Sugars and other signals can affect the localization of
sucrose synthase, providing a potential means of finetuning the balance between respiration and biosynthesis
[2,71,85]. Although sucrose synthase has a non-specific
affinity for membranes [85], early work indicated that the
phosphorylation status of the enzyme is involved in
regulating this association [57,72]. This is further supported by the sensitivity of candidate kinases to cellular
signals [49,75,86–89]. The extent of sucrose synthase
phosphorylation in different fractions has varied with
studies and/or systems, however, such that the free cytoplasmic enzyme can be hypo- [72,73], hyper- [72,73], or
equally phosphorylated [89] relative to membrane- or
actin-bound forms. Emerging data indicate that developmental status can influence this relationship [85], possibly because of shifts in membrane type or composition.
Actin
Regulation of sucrose synthase protein turnover
Sucrose
synthase
Cell wall
biosynthesis
(Special functions)
(c)
Cytoplasmic
Membrane
-bound
Plasma membrane
Golgi
SUS
(Respiration)
influence the balance between sucrose synthase’s role in
respiration and special functions in biosynthesis or compartmentalization (Figure 5c). The enzyme is typically
soluble in the cytoplasm and can contribute readily to an
ATP-conserving path of respiration (Figure 4). However,
it can also move rapidly on and off membrane or cytoskeletal locations, indicating that it might also have a
number of special functions. These include activities at
plasma membrane and Golgi sites for cell wall biosynthesis [39,51,81–83], a possible role at the tonoplast that is
related to the use and/or storage of vacuolar sucrose [84],
and an activity at points on actin that could facilitate
starch formation through plastid proximity and/or other
metabolic shuttling via ‘metabalons’ [2,71,72].
Tonoplast
Vacuole
Current Opinion in Plant Biology
Regulation of sucrose synthase through sub-cellular localization. (a) A
rapidly growing body of evidence supports an association between
sucrose synthase and rosettes of the plasma membrane cellulose
synthase complex, much as originally proposed by Delmer and
coworkers [82]. (b) The biochemical features of this model facilitate the
direct transfer of UDPG from the sucrose synthase reaction into
cellulose biosynthesis, and the rapid recycling of UDP. (c) Recent
work has indicated a broad spectrum of apparently transient
associations of sucrose synthase with membranes and/or actin. The
mechanisms that regulate this change in locale have become a
central issue, because they could provide a means of controlling the
balance between the cytoplasmic respiratory functions of sucrose
synthase and special roles of this enzyme in other processes. Among
the latter processes are cell wall biosynthesis (in the plasma
membrane and Golgi), starch biosynthesis (in the plastids), metabolic
channeling via metabalons (an actin-related process), and possibly
compartmentalization and/or mobilization (involving the tonoplast or the
sieve-tube plasma membrane). Cellular sugar status, developmental
stage, and phosphorylation appear to influence the shift of sucrose
synthase from cytoplasmic to membrane-bound forms.
Current Opinion in Plant Biology 2004, 7:235–246
Despite the ‘extreme stability’ of sucrose synthase protein under some conditions [77], it is becoming apparent
that several mechanisms contribute to tightly control its
turnover (Figure 6). First, the phosphorylation that activates the enzyme (at the S15 site or its equivalent) [86,87]
is also implicated in predisposing sucrose synthase to
phosphorylation at a second site (S170 or its equivalent)
[90]. This second phosphorylation, in turn, targets the
protein for ubiquitin-mediated degradation via the proteosome [90]. Phosphorylation at the first site can be
linked to sugar availability and/or to other signals through
the catalysis of this step by both Ca2þ-dependant protein
kinases (CDPKs) [87,90,91] and/or a sugar-responsive
Ca2þ-independent SnRK [75,88,89,90]. The second
phosphorylation can be catalyzed by CDPKs but not
SnRKs [90]. Second, this avenue of sucrose synthase
breakdown can be inhibited if the second phosphorylation site (S170 or equivalent) is blocked by the binding of
ENOD40 proteins (which were initially named for their
role in early nodule development but which are now
known to have diverse functions throughout the plant)
[90,92,93]. The protective association of ENOD40s
with sucrose synthase has been implicated in the control
of vascular function, phloem loading/unloading, and
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Sucrose metabolism Koch 241
Figure 6
Stable SUS
S15 S170
SUS
Activated
by +P
P
P
S15 S170
S15
ENOD
proteins
S170
SUS
SUS
Unstable SUS
P
P
P
S15
S170
+ P by CDPKs
SUS
P
P
S15 S170
Ubiquination and
degradation by proteosomes
SUS
the primary path of cleavage for sucrose entering growing/
expanding sink tissues. The transfer of sucrose to
vacuoles for initial metabolism has a dual advantage in,
first, giving a two-for-one return on the cost of transporting osmotically active solutes into the vacuole for expansion and, second, adjusting organ expansion relative to
carbohydrate supply. Such contributions by vacuolar
invertase depend, however, on continued capacity for
cell wall expansion. If turgor exceeds this potential, then
sucrose cleavage by these invertases could have a minimal
or even a negative effect on sucrose import [24]. During
sink initiation and the initial expansion growth of many
sinks, however, vacuolar invertases appear to have a key
role [5,9,35,36,95].
Vacuolar invertase expression is also sensitive to an array
of signals, including sugars, hormones, and environmental
stimuli [3,13,14,17,96]. Hence, these enzymes are in a
position to modulate responses to diverse inputs. Among
these are the influence of gravity and indole acetic acid
(IAA) on bending stems [13], cytokinins and IAA on the
formation and expansion of tumors [17], drought and
ABA on hexose levels in leaves [14], and cold on the
sweetening of tubers [96]. The apparent contribution of
vacuolar invertase in the adjustment of reproductive load
under stress and resource limitation, when the early
abortion of some fruits or seeds can allow the survival
of others, is also significant [36].
Current Opinion in Plant Biology
Roles of cell wall invertases
Current model for SUS regulation through protein turnover. (a) Stable
forms of SUS include those activated by phosphorylation at an S15
site (or equivalent) [49,73,86,87], especially if a second phosphorylation
site is blocked by the binding of ENOD proteins [90]. (b) If the protective
ENOD protein is absent, however, the initial phosphorylation event
predisposes SUS to a degradative process that begins with a second
phosphorylation at the S170 site (or its equivalent). This targets SUS
for ubiquitin-mediated degradation by the proteosome [90]. The
protective association of ENOD40s with sucrose synthase has been
implicated in control of vascular function, phloem loading/unloading,
and assimilate import [92,93,94]. P, phosphate; S, serine.
assimilate import [90,92,94]. ENOD40 mRNAs are
elevated, for example, at sites of high sink activity and
at points of rapid unloading in phloem.
Roles and regulation of invertases
Roles of vacuolar invertases
It is now becoming evident that vacuolar invertases contribute prominently to both sucrose import ([5,17,36];
P Commuri et al., unpublished data) and sugar signals
[9,27,32], particularly during the expansion growth of
diverse sink structures [1,5,9,17,26,36,95]. These roles
for vacuolar invertases are additional to previously recognized functions including turgor regulation and the control of sugar balance in fruit tissues and mature tubers
[5,9,96]. The sucrose import and potential signaling
influences of vacuolar invertases arise from their role in
www.sciencedirect.com
Cell wall invertases are central to phloem unloading in
some, but not all, sucrose-importing structures. Their
significance is most prominent in sinks in which an
apoplastic (cell wall) step is involved because of a gap
in plasmodesmatal connections between cells [9,25]. This
occurs in developing seeds and pollen, where an unloading role for cell wall invertases is consistent with results
from Vicia faba [6], barley [26], and maize [37]. Stress to
ovaries during early development can also induce abortion, which is preceded by rapid changes in first vacuolar
then cell wall invertases ([36]; P Commuri et al., pers.
comm.; J Mullin, pers. comm.). Cell wall invertase contributes predominantly to the development of pollen
(another apoplastically isolated sink structure), and localized antisense reductions in cell wall invertases can be
used to manipulate male fertility [16]. Cell wall invertases
can also influence sinks in which plasmodesmatal connections remain intact, if at least some sucrose moves
across the cell wall space. There is evidence in support of
such a role for cell wall invertases in developing carrot
roots [5], potato tubers [48], and in response to signals
from some biotic [17,27] and abiotic stresses [5].
The highly responsive transcriptional regulation of
invertases
Invertase genes respond to diverse signals, including
those generated by direct and indirect effects of their
Current Opinion in Plant Biology 2004, 7:235–246
242 Physiology and metabolism
own expression [3,15,16]. Invertases are also repressed
by low oxygen, strongly and rapidly enough to serve as
potential markers of endogenous oxygen availability
[7]. In addition, invertases respond to a full spectrum
of plant hormones, and to a wide range of environmental
and pathogenic signals [13,14,15,16]. Different family
members can also show contrasting responses to sugars or
plant hormones [3,14,36], and the expression of the
same gene can differ markedly with tissue and/or conditions. The maize Ivr2 vacuolar invertase gene, for example, is upregulated in the leaves of drought-stressed
plants [14] but downregulated in ovaries and young
kernels under similar conditions [36]. The effects of
stress signals, including ABA ([14]; K Koch, unpublished), are modified by other developmental signals.
Collectively, these signals facilitate the contributions of
invertase to the survival and acclimation of leaves, yet
restrict reproductive investment to a limited number of
offspring that will have a greater chance of support to
maturity.
Regulation of targeting and turnover for vacuolar
invertase
The regulation of vacuolar invertase at the protein level
involves a previously unrecognized means of controlling
both compartmentalization and breakdown, the precursor
protease vesicle (PPV) and vacuolar processing enzyme
(VPEg) system ([97]; Figure 6). Analysis of an Arabidopsis vacuolar invertase (AtFRUCT4 [At1g12240]) shows
that this protein can be compartmentalized for extended
periods in the PPVs, which are distinctive spindle-shaped
endomembrane vesicles that lie between ribosomes and
vacuoles [97]. This storage of invertases can apparently
delay delivery to vacuoles, thus imposing an additional
level of control beyond that of mRNA-, protein-, or totalenzyme activity levels. In addition, these PPV compartments house an inactive form of VPEg protease
(At4g32940) that can be released into vacuoles together
with the invertase, and that can subsequently target the
invertase for degradation [97]. The VPEg auto-activates
upon entry into the acidic vacuole, and includes at least
one vacuolar invertase among its specific substrates. The
PPV compartmentalization of a vacuolar invertase could
thus regulate both the time at which its activity in
vacuoles begins and its vulnerability to subsequent turnover by the VPEg protease (Figure 7).
Regulation of invertases by inhibitor proteins
Inhibitor proteins are gaining increasing recognition as a
potentially important means of in-vivo control for invertase activity. The presence of these relatively small
protein inhibitors has been well documented in various
systems, and they inhibit both vacuolar and cell wall
invertases when present in extracts. However, the sites
of action and functional significance of these proteins in
vivo have not been fully defined. Although most inhibitor
proteins appear to be cell wall proteins, a putative vacuolar homolog of a cell wall form from tobacco has been
identified and introduced into transgenic potatoes [96].
The tubers of these plants had reduced capacity for the
cold-induced sweetening that typically results from hexose accumulation in vacuoles. A probable effect within
the vacuole was indicated for this transgenic system. In
addition, the recent work of Bate et al. [98] demonstrated
Figure 7
Vacuole
Ribosomes
PPV
VIN
active?
VPEγ
inactive
VIN
VIN transfer and extended
transcription
protective storage
VIN
active
VIN
degraded
VPEγ
auto-activated
VIN turnover by the VPEγ
cysteine protease system
Current Opinion in Plant Biology
Regulation of vacuolar invertase (VIN) at the protein level. The transfer, protective retention, and protein turnover of VIN are potentially modulated by
the precursor protease vesicle (PPV) and vacuolar processing enzyme (VPEg) system. At least some of the newly translated VIN enters the PPV,
where it can be retained for extended periods. It is presumably protected from proteolytic degradation in this compartment, but its functional
role remains unclear until it enters the vacuole. The PPV also stores a VPEg protease, which remains inactive until it too moves into the vacuole.
Once activated, VPEg targets specific substrates that include a VIN. This invertase may thus be targeted for degradation as soon as it enters the
site of its presumed action in vivo. The PPV and VPEg system therefore provides a potential mechanism for modifying both the timing and the
duration of at least some VIN activity.
Current Opinion in Plant Biology 2004, 7:235–246
www.sciencedirect.com
Sucrose metabolism Koch 243
that a maize invertase inhibitor, ZmINVINH1, is exported to the apoplast, where it could interact with invertases
during early kernel development (i.e. 4–7 days after
pollination). These researchers further defined the most
prominent site of ZmINVINH1 expression as the embryo
surrounding region (ESR), which they suggest may help
to preserve crucial differences in the extracellular sugar
environments of the embryo and endosperm during early
development. Greater hexose levels in the endosperm
apoplast may favor more rapid cell division and minimal
differentiation relative to those in embryos [6].
Conclusions
Sucrose-cleaving enzymes lie at the heart of mechanisms
for the distribution and use of sucrose within multicellular
plants. They also occupy a pivotal position in the balance
between the different sugar signals generated by
imported sucrose. Their regulation has thus become
the focus of considerable interest, and involves diverse
and highly integrated mechanisms operating at transcriptional and posttranscriptional levels.
Acknowledgements
Supported by the US National Science Foundation (Cellular
Biochemistry) and by the Florida Agricultural Experiment Station
(Journal Series Number R-10079).
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