sucrose metabolism: regulatory mechanisms and...

Sucrose metabolism: regulatory mechanisms and pivotal rolesin sugar sensing and plant developmentKaren 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.

AddressesPlant Molecular and Cellular Biology Program, Horticultural Sciences

Department, University of Florida, Gainesville, Florida 32611, USA

e-mail: [email protected]

Current Opinion in Plant Biology 2004, 7:235–246

This review comes from a themed issue on

Physiology and metabolismEdited by Christoph Benning and Mark Stitt

1369-5266/$ – see front matter

� 2004 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2004.03.014

AbbreviationsABA abscisic acid

CIN cytoplasmic invertase

CWIN cell wall invertase

ENOD early nodulationPPi inorganic pyrophosphate

PPV precursor protease vesicle

SnRK SNF-related kinase

SUS sucrose synthase

UDP uridine di-phosphate

UDPG UDPglucose

VIN vacuolar invertase

VPEc vacuolar processing enzyme g

IntroductionThe only known enzymatic paths of sucrose cleavage in

plants are catalyzed by invertases (sucrose þ H2O !glucose þ fructose) and sucrose synthases (sucrose þUDP ! 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 biosynth-

esis 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 develop-

mental system in plants. Its influence appears to be

balanced by the capacity for sensing sucrose itself,

Figure 1

Current Opinion in Plant Biology

Invertase

Glucose

FructoseHexosesignals

?

Fructose

Hexose signals

Sucrose signals

Sucrose synthase

UDP glucose

Sucrose

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 overallsubstrate 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.

www.sciencedirect.com Current Opinion in Plant Biology 2004, 7:235–246

but different systems and responses are involved

[18,19�,20�,21,22�].

Additional factors in the link between sucrose metabo-

lism 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 con-

nections 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

these instances can favor sucrose import and amplification

of hexose signals if sucrose moves out into the extracel-

lular 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 pro-

ducts 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 inver-

tases (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 counter-

parts, 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 plantdevelopment through sugar signalsIn 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 develop-

ment [6,26�,33,34]. According to this hypothesis, inver-

tases mediate the initiation and expansion of many new

sink structures [5,9,35], often with vacuolar activity pre-

ceding that in cell walls [17�,36�]. The action of cell wall

invertase coincides with the elevated expression of hex-

ose 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 sucrose-

synthase paths of sucrose cleavage. In some highly loca-

lized 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 devel-

opment (e.g. in the phloem and at sites of rapid cell wall

Figure 2


Vacuole

G + F UDPG + F G + F

Sink cell cytoplasm

Sucrose

Phloem

Sucrose

Cell wallspace

Plasmo-desmata

G + F

CIN SUS VINCWIN

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 otherhexoses 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.

236 Physiology and metabolism

Current Opinion in Plant Biology 2004, 7:235–246 www.sciencedirect.com

deposition [17�,38–40]). Overall, however, diverse evi-

dence supports the contention that the balance between

invertase and sucrose synthase activity can alter plant

development through differential effects on sugar signal-

ing systems.

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 post-

transcriptional 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 strik-

ing 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 meta-

bolism by those hormone systems together comprise a

means of integrating individual cellular responses into

those of the multicellular organism.

Roles and regulation of sucrose synthasesEssential 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 high-

cellulose 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 reduc-

tions 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

Figure 3


Rel

ativ

e co

ntrib

utio

nto

suc

rose

met

abol

ism

Sink initiation and expansionMaximal generation of vacuolar hexoses- Expansion (linked to photosynthate availability)- Respiration- Hexose-based sugar sensing (cell division and initial differentiation)

Vacuolar invertases:

Cell wall invertases:

Continued sink initiation and expansionMaximal 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)

Sucrose synthases:

Storage and maturationGeneration 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)

Vacuolarinvertases

Cell wallinvertases

Sucrosesynthases

Developmental progression

A common developmental profile of the contributions of sucrose-cleaving 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 ATP-

conserving 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 sinkdevelopment.

Sucrose metabolism Koch 237


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 exacer-

bate the problem of oxygen depletion [53�]. Evidence

from transgenic potatoes shows that the normal develop-

mental shift to sucrose cleavage by sucrose synthase in

wildtype tubers improves not only their adenylate bal-

ance, 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 sur-

vive flooding [56,57]. Under pronounced low-oxygen

conditions, sucrose synthase responds rapidly to early

rises in cytosolic Ca2þ [31,50] and can support consider-

able biosynthesis (of cellulose and callose) [50,51]. The

functional significance of sucrose synthase, as opposed to

invertase, is likely to be particularly important in low-

oxygen conditions where it can aid the respiratory con-

servation 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 trans-

genically are also ultimately smaller than those of wild-

type plants [5], and similar tomato transgenics showed

reduced fruit set from the earliest flowers [64]. A sucrose

synthase path of sucrose cleavage also typically predomi-

nates 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

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 trans-

ferase 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 inor-

ganic pyrophosphate (PPi) is essential for phloem func-

tion, 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 sieve-

tube locale could also facilitate sucrose synthase’s prob-

able 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 avail-

ability. Those sucrose synthase genes that are upreg-

ulated by carbohydrate deprivation are also the most

strongly induced by low-oxygen, and show narrow pat-

terns of expression that could confer sucrose import or

survival priority for key tissues [3,8,30]. The starvation-

inducible 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



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 involve-

ment in the induction of rice Rsus1 [77], separate roles for

both hexokinase-dependent and -independent sugar-

sensing mechanisms are implicated in the responses of

an Arabidopsis Sus1 [78].

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 145-

bp 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].

Translational control of sucrose synthase

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 en-

hanced 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 posttranscrip-

tional levels may have specific physiological significance.

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 com-

plex (Figure 5). Such a relationship could allow UDPG

from sucrose synthase to feed directly into cellulose for-

mation, 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

Figure 4


Sucrose synthaseSieve-tube element

Companion cell

Openlumen

Cellwall

UDP

UDPG Fructose

UTP

G1P

UGPase

Callosesynthase

PPiPPi

F6P

Sieve-tube

lumen

Sieve-tube cytoplasm

Biosynthesis Respiration

Sucrose

Sucrose synthase

H+

Glycolysis

?

?

Sucrose

ATP

H+

ADP

ATPfor transporter

Callosefor plugging

(a)

(b)

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

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-1-

phosphate.



influence the balance between sucrose synthase’s role in

respiration and special functions in biosynthesis or com-

partmentalization (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 cyto-

skeletal locations, indicating that it might also have a

number of special functions. These include activities at

plasma membrane and Golgi sites for cell wall biosynth-

esis [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].

Sugars and other signals can affect the localization of

sucrose synthase, providing a potential means of fine-

tuning 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 sup-

ported 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 cyto-

plasmic enzyme can be hypo- [72,73�], hyper- [72,73�], or

equally phosphorylated [89] relative to membrane- or

actin-bound forms. Emerging data indicate that develop-

mental status can influence this relationship [85�], possi-

bly because of shifts in membrane type or composition.

Regulation of sucrose synthase protein turnover

Despite the ‘extreme stability’ of sucrose synthase pro-

tein under some conditions [77], it is becoming apparent

that several mechanisms contribute to tightly control its

turnover (Figure 6). First, the phosphorylation that acti-

vates 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 pro-

teosome [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 phosphoryla-

tion 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

Figure 5


(c)

(a)

Sucrose synthase

Cellulose synthase complex

Cel

lulo

se

Sucrosesynthase

Cellulosesynthase

Cellulose

Fructose

UDP

UDPGUDPG

UDP

Cell wall

Poresubunit

Plasma membrane

Sucrose

Sucrose

(b)

Plasmamembrane

Cell wall

Cytoplasm

Cytoplasm

Cell wallbiosynthesis

SUS

CytoplasmicMembrane-bound

(Respiration)

(Special functions)

Vacuole

Plasma membrane

Golgi

Amyloplasts

Actin

Tonoplast

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 plasmamembrane 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.



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 invertasesRoles of vacuolar invertases

It is now becoming evident that vacuolar invertases con-

tribute 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 recog-

nized functions including turgor regulation and the con-

trol 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

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 transport-

ing osmotically active solutes into the vacuole for expan-

sion 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�].

Roles of cell wall invertases

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 unload-

ing 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 abor-

tion, 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 con-

tributes predominantly to the development of pollen

(another apoplastically isolated sink structure), and loca-

lized antisense reductions in cell wall invertases can be

used to manipulate male fertility [16]. Cell wall invertases

can also influence sinks in which plasmodesmatal con-

nections 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

Figure 6


S15 S170

SUS

S15 S170

SUS

Stable SUS

S15 S170

SUS

ENODproteinsP P

Unstable SUS

Ubiquination anddegradation by proteosomes

S15 S170

SUS

P P

S15 S170

P P

SUS

P

+ P by CDPKs

Activatedby +P

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.



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 condi-

tions. The maize Ivr2 vacuolar invertase gene, for exam-

ple, 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, unpub-

lished), 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 Arabidop-sis 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 total-

enzyme activity levels. In addition, these PPV compart-

ments 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 turn-

over 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 inver-

tase 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 invivo have not been fully defined. Although most inhibitor

proteins appear to be cell wall proteins, a putative vacuo-

lar 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 hex-

ose 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


Ribosomes

PPV

VINtranscription

VIN transfer and extendedprotective storage

VIN turnover by the VPEγcysteine protease system

Vacuole

VPEγauto-activated

VINdegraded

VINactiveVIN

active?

VPEγinactive

Regulation of vacuolar invertase (VIN) at the protein level. The transfer, protective retention, and protein turnover of VIN are potentially modulated bythe 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.



that a maize invertase inhibitor, ZmINVINH1, is export-

ed 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].

ConclusionsSucrose-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 transcrip-

tional and posttranscriptional levels.

AcknowledgementsSupported by the US National Science Foundation (CellularBiochemistry) and by the Florida Agricultural Experiment Station(Journal Series Number R-10079).

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

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88. Chikano H, Ogawa M, Ikeda Y, Koizumi N, Kusano T, Sano H: Twonovel genes encoding SNF-1 related protein kinases fromArabidopsis thaliana: differential accumulation of AtSR1 andAtSR2 transcripts in response to cytokinins and sugars, andphosphorylation of sucrose synthase by AtSR2. Mol GenGenet 2001, 264:674-681.

89. Haigler CH, Ivanova-Datcheva M, Hogan PS, Salnikov VV,Hwang S, Martin K, Delmer DP: Carbon partitioning tocellulose synthesis. Plant Mol Biol 2001, 47:29-51.



90.�

Hardin SC, Tang G-Q, Scholz A, Holtgraewe D, Winter H, Huber SC:Phosphorylation of sucrose synthase at serine 170: occurrenceand possible role as a signal for proteolysis. Plant J 2003,35:588-603.

The authors describe a previously unrecognized phosphorylation site ofsucrose synthase and its potential role in controlling the turnover of thesucrose synthase protein.

91. Cheng SH, Willmann MR, Chen HC, Sheen J: Calcium signalingthrough protein kinases. The Arabidopsis calcium-dependentprotein kinase gene family. Plant Physiol 2002, 129:469-485.

92. Kouchi H, Takane K, So RB, Ladha JK, Reddy PM: Rice ENOD40:isolation and expression analysis in rice and transgenicsoybean root nodules. Plant J 1999, 18:121-129.

93.�

Rohrig H, Schmidt J, Miklashevichs E, Schell J, John M: SoybeanENOD40 encodes two peptides that bind to sucrose synthase.Proc Natl Acad Sci USA 2002, 99:1915-1920.

A combination of in-vitro translation and western blotting indicates thattwo small ENOD40 peptides are produced from overlapping regions atthe 50 end of the same mRNA. Further work demonstrates that thesepeptides bind specifically to sucrose synthase. This work, together withdata from other studies [90�,94�], implicates these ENOD40 peptides inenhancing the activity and longevity of sucrose synthase in diversesystems.

94.�

Varkonyi-Gasic E, White DW: The white clover enod40 genefamily. Expression patterns of two types of genes indicate arole in vascular function. Plant Physiol 2002, 129:1107-1118.

In-situ localization of clover ENOD40 mRNAs showed them to be presentin vascular regions, especially at sites of extensive sucrose transfer.Together with data from [90�], this work indicates a role for ENOD40 instabilizing functional sucrose synthase at these sites.

95. Klan EM, Hall B, Bennett AB: Antisense acid invertase (TINV1)gene alters soluble sugar composition and size in transgenictomato fruit. Plant Physiol 1996, 112:1321-1330.

96. Greiner S, Rausch T, Sonnewald U, Herbers K: Ectopic expressionof a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers. Nat Biotechnol 1999,17:708-711.

97.�

Rojo E, Zouhar J, Carter C, Kovaleva V, Raikhel NV: A uniquemechanism for protein processing and degradation inArabidopsis thaliana. Proc Natl Acad Sci USA 2003,100:7389-7394.

The authors describe a mechanism for the protective storage of vacuolarinvertase and a possible delay in its vacuolar action. A system for theinactivation of this enzyme after its release into the vacuole is alsodescribed (and involves proteolysis by a specific VPEg).

98.�

Bate NJ, Niu X, Wang Y, Reimann KS, Helentjaris TG: An invertaseinhibitor from maize localizes to the embryo surroundingregion during early kernel development. Plant Physiol 2003,134:1-9.

Several lines of evidence indicate that a localized expression of a cell wallinvertase inhibitor from maize may have implications for early embryodevelopment.



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