Sucrose Transport in Higher Plants

Article (PDF Available)inInternational Review of Cytology 178:41-71 · February 1998with54 Reads
DOI: 10.1016/S0074-7696(08)62135-X · Source: PubMed
Presumably due to its physicochemical properties, sucrose represents the major transport form of photosynthetically assimilated carbohydrates in plants. Sucrose synthesized in green leaves is transported via the phloem, the long distance distribution network for assimilates in order to supply nonphotosynthetic organs with energy and carbon skeletons. At least in Solanaceae, sugar export seems to be a tightly regulated process involving a number of specific plasma membrane proteins. Significant progress in this field was made possible by the recent identification of plasma membrane sucrose transporter genes. These carriers represent important parts of the long-distance transport machinery and can serve as a starting point to obtain a complete picture of long-distance sucrose transport in plants. A combination of biochemical studies of transporter properties together with expression and localization studies allow specific functions to be assigned to the individual proteins. Furthermore, the use of transgenic plants specifically impaired in sucrose transporter expression has provided strong evidence that SUT1 transporter function is required for phloem loading. Physiological analyses of these plants demonstrate that sucrose transporters are essential components of the sucrose translocation pathway at least in potato and tobacco.


Journal of Experimental Botany, Vol. 50, Special Issue, pp. 935 –953, June 1999
Update on sucrose transport in higher plants
Christina Ku
¨hn1, Laurence Barker, Lukas Bu
¨rkle and Wolf-Bernd Frommer
Pflanzenphysiologie, ZMBP–Centre of Plant Molecular Biology, Universita
¨bingen, Auf der Morgenstelle 1,
D-72076 Tu
¨bingen, Germany
Received 10 August 1998; Accepted 30 November 1998
of water and mineral salts taken up by the root system
and transported via the xylem network, while non-green
Sucrose as the major transported form of fixed carbon, tissues depend on the carbon supply deriving from meta-
must be translocated from the sites of synthesis, i.e. bolic source organs via the phloem. The existence of a
the green tissues, to the sites of consumption and well co-ordinated long-distance transport network was a
storage, i.e. the non-green cells and organs. For apo- prerequisite for the evolutionary development of higher
plasmic transport, carrier-mediated processes are land plants. To understand carbon allocation throughout
required at the plasma membrane. Functional comple- the whole plant, it is necessary to describe the phloem
mentation of modified yeast strains has enabled the network in more detail ( Behnke, 1989).
isolation and characterization of a large family of suc-
rose carriers (SUT) from a wide variety of species.
The route of photosynthetic carbon through the plant
In Xenopus oocytes, electrophysiological methods
demonstrated that the SUTs function as proton Fixation of CO2takes place in the chloroplasts of meso-
co-transporters. Localization studies show that at phyll cells, mainly in the palisade parenchyma of mature
least SUT1 is present at the plasma membrane of leaves by the Calvin cycle. Net products of this cycle are
enucleated sieve elements, indicating macromolecular triose phosphates which can be fed into several biosyn-
transport of its mRNA or protein from the companion thetic pathways, e.g. starch, lipid or amino acid biosyn-
cell to the sieve element. Inhibition of the transport thesis in chloroplasts or sucrose and amino acid synthesis
activity in several transgenic plant species proves that in the cytosol. Various biosynthetic pathways in dierent
SUT function is essential for long-distance transpor t. compartments of mesophyll cells compete with each other
Further experiments will be required to assign specific for triose phosphates (Schulz et al., 1993 ). According to
functions to the other members of the SUT family. the requirements of the whole plant, the direction of
biosynthesis is determined by the exchange rate of triose
Key words: Sucrose, transport, sucrose carriers, proton phosphates across the inner chloroplastidic membrane
co-transporters, plasma membrane, sieve elements, com- which depends on the transport activity of the T riose-
panion cells. P hosphate-Translocator ( TPT ) located in the inner envel-
ope. First identified in spinach leaves as a 29 kDa protein,
the TPT works as an antiporter mediating the export of
3-phosphoglycerate as triose phosphates from the chloro-
plast matrix in exchange for inorganic phosphate from
In contrast to unicellular organisms such as bacteria,
the cytosol (Flu
¨gge et al., 1989). The availability of
many fungi and unicellular green algae, higher plants are
inorganic phosphate depends mainly on cytosolic sucrose
able to develop highly dierentiated tissues that fulfil
synthesis, which releases inorganic phosphate in several
specific functions. These specializations accompany a loss
steps. In simple terms, a decrease in sucrose synthesis
in autonomy making it indispensable for dierent cell
therefore leads to reduced levels of inorganic phosphate,
types to collaborate closely with one another even over
thus down-regulating triose phosphate export, which leads
long distances. Phloem and xylem elements form a capil-
to the deposition of transitory starch in the chloroplast.
lary network connecting metabolic sink and source
organs. Thus green tissues are dependent on the supply The TPT appears to be a well-regulated membrane protein
1To whom correspondence should be addressed. E-mail:
© Oxford University Press 1999
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936 Ku
¨hn et al.
allowing for the adaptation of leaf carbon metabolism to The nucleus disintegrates either by pycnosis or by
chromatolysis (Behnke and Sjo
¨lund, 1990; Eleftheriou,the momentary requirements for transient storage or
long-distance transport of photosynthates ( Flu
¨gge et al., 1990; Evert, 1990) and the tonoplast undergoes selective
autophagy. The fragments of the tonoplast membrane1989).
In most plants, sucrose is the main transport form of associate with other remnants of the endomembrane
system and are assumed to reorganize to form the parietal,photoassimilates, in contrast to hexoses that do not
circulate over long distances as in the animal kingdom. agranular SE-specific sER, which is seemingly free of
ribosomes. The hypothesis of reorganization needs furtherSucrose moving from source to sink organs has to over-
come several membranes involving specific sucrose car- confirmation by localization of tonoplast-specific proteins
in the sER membrane system in mature SEs.riers. This paper focuses on this particular group of
membrane transporters in vascular land plants. In SEs, two types of plastids can be observed: the
so-called S-plastids containing large starch granules and
the P-plastids with proteinaceous inclusions which are
Phloem anatomy
present in parietal positions in SEs. The starch granules
present in S-plastids of SEs are spherically shaped andPhloem and xylem form collateral bundles. The pro-
tophloem elements arise from procambial strands and are rich in amylopectin. The content of P-plastids may play
a role in sieve pore plugging (‘two component glue’, seeonly functional in proximity to meristematic apices.
Functionality is then taken over by the metaphloem Knoblauch and van Bel, 1998 ). Mature SEs are sur-
rounded by a plasma membrane and are considered toelements within and beyond the extension zone. Meta-
phloem elements arise from procambial tissue, or in the be living cells, still having the capacity to plasmolyse in
hyperosmotic media, although they do not have of secondary phloem elements, from the cambium
(Esau, 1969; Behnke, 1989). Deprived of essential organelles, SEs lose their autonomy
during maturation, suggesting that one important task ofThe phloem of angiosperms comprises several cell types:
the phloem sap conducting sieve elements (SEs), the the CCs’ numerous ribosomes and mitochondria is the
supply of compounds to the highly specialized SEs (for acompanion cells (CCs) and the phloem parenchyma cells.
In their mature form, SEs are interconnected via sieve review see Sjo
¨lund, 1997).
The ultrastructure of phloem cells appears to reflectpores at their terminal ends. SEs and CCs develop by
asymmetric cell division from a common mother cell. In the function of these cells. Thus, in the past, several
anatomical features of phloem cells have been correlatedcontrast to the sieve tube member, the CC retains its
capacity for further divisions or redierentiation. During with dierent phloem functions, such as phloem loading,
unloading, or retrieval of translocated assimilates.maturation, SEs undergo selective autophagy, and the
plasmodesmata (PD) at the poles widen to form sieve A further ultrastructural hint for the specific task
performed by phloem cells comes from freeze–fracturepores. Sieve pores arise during cytokinesis from primary
PD through cell wall disintegration. The resulting large analysis of Streptanthus callus SE plasma membranes.
The internal and external face of phloem cell plasmapores allow free movement of solutes in the phloem
translocation stream through sieve plates. The ER cis- membranes show diering densities of intramembraneous
particles (IMP). The distribution of IMP within the twoternae play an important role in the formation of sieve
pores in dicotyledons, as does the deposition of callose. membrane faces diers between SEs and parenchyma
cells. In parenchyma cells, the ratio of IMP at theDuring cell division of their common mother cell, SEs
and CCs remain in close symplasmic contact by numerous cytoplasmic (P-face) to that of the external ( E-face) is
approximately 1051(PF5EF ). This relationship is shiftedasymmetrically branched PD and behave as a single
functional unit, which has led to the term sieve element– to about 151 in SEs. The high particle density of the SE
E-face seems to be a morphological manifestation ofcompanion cell complex (SECCC). During SE dierenti-
ation the cell wall thickens and autolytic degradation intensive active transport across the plasma membrane of
SEs. IMPs are thought to be the sites of proton-pumpingprocesses break down a large part of the SE cytoplasmic
content such as nucleus, tonoplast, dictyosomes, and most ATPases and specific carriers for sucrose or amino acids
¨lund and Shih, 1983a).of the ribosomes. Cell wall thickening of the SE could be
a prerequisite necessary for the increased turgor pressure The postulation that, in some species, phloem loading
occurs directly at the plasma membrane is supported byduring active translocation in mature SEs. Sieve areas in
lateral and terminal walls (sieve plates) do not undergo the anatomy of the modified ER in SEs. A modified
agranular ER resides as a parietal anastomosing system.cell wall thickening. In mature SEs, only a few plastids,
mitochondria and a modified ER persist, mostly in pari- The function of the close spatial relationship between
plasma membrane and ER is thought to provide a micro-etal positions. There is experimental evidence that mito-
chondria are metabolically active even in mature SEs environment for proton-eux and proton/sucrose
cotransport through the plasma membrane, spatially sep-(McGivern, 1957; Lee et al., 1971; Moniger et al., 1993 ).
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Sucrose transport in higher plants 937
arated from the rapid translocation stream in the SE still unknown how they are synthesized and metabolized.
The structural P-proteins, major components of thelumen (Sjo
¨lund and Shih, 1983b). For a recent review
about the relationship between phloem anatomy and phloem exudate in dicotyledons have been shown to
be stable, at least over a 72 h period in cucurbitsfunction, see Schulz (1998 ).
(Dammenhofer et al., 1997). However, as evidenced by
enzyme activities, the protein concentration in exudates
of non-cucurbits remains constant over many hours of
Sieve element proteins
exudation (Becker et al., 1971). The question is, where
Besides ions and metabolites, the phloem sap contains a the synthesis of SE specific proteins takes place? It is
distinct set of SE-specific proteins. Using the insect laser likely that these proteins are synthesized in CCs and
technique, Nakamura et al. (1993) collected a set of subsequently translocated via PD into the SE. This hypo-
proteins from the rice phloem sap, the composition of thesis is supported by the finding that P-protein PP2
which remained stable over a 6 d period. This surprising mRNA is found in CCs, whereas PP2 protein is present
finding suggests that some of the SE proteins are subject in SEs and CCs (Bostwick et al., 1992; Dannenhofer et
to constant de novo synthesis. The most abundant of the al., 1997 ). Synthesis of P-proteins from Cucurbita maxima
phloem proteins are the structural P-proteins occurring occurs in CCs, as shown by microautoradiography
in either crystalline (P-protein bodies) or filamentous (Nuske and Eschrich, 1976).
form (P-protein tubules and filaments) in the SEs Most of the phloem proteins are probably synthesized
(40–100 mgmg1phloem exudate in Cucurbitaceae in CCs and tracked through PD into the SEs. It has
(Eschrich, 1975). P-protein filaments, which may play a been shown that several phloem proteins from Ricinus
role in plugging sieve pores, are composed of PP1, a communis and Cucurbita maxima, e.g. glutaredoxin and
filamentous protein of 90–100 kDa, and PP2, a phloem cystatin, are able to interact with mesophyll PD to
lectin of 23–28 kDa with haemagglutinating properties, increase their size exclusion limit (SEL) (Balachandran
that binds to PP1 as a dimer (for review, see Cronshaw et al., 1997 ). Surprisingly, Ishiwatari and coworkers were
and Sabnis, 1990). PP2 mRNA from cucurbits has been able to identify thioredoxin h as the major protein in the
localized by RNA in situ hybridization exclusively in CCs, rice phloem sap, probably due to the absence of p-
whereas PP2 protein has been detected in both SEs and proteins from many monocots (Ishiwatari et al., 1995).
CCs (Bostwick et al., 1992). P-proteins are reportedly absent from many monocots
The identification of structural phloem proteins was (Cronshaw, 1981 ). Co-injection experiments in rice with
successful by the use of monoclonal antibodies against fluorescently labelled dextrans demonstrated that thiore-
purified SEs isolated from Streptanthus callus cultures doxin h can mediate its own cell-to-cell transport through
¨lund, 1990). One of the antibodies interacts specific- PD by modifying the plasmodesmal SEL values to greater
ally with a SE specific b-amylase of 57 kDa ( Wang et al., than 9.4 kDa, ( Ishiwatari et al., 1998 ). It has been argued
1995a). Antibodies against P-proteins also gave positive that this reductase could play a role in repair of other
signals (Toth and Sjo
¨lund, 1994; Toth et al., 1994). phloem proteins (Raven, 1991), or as a regulator of
Soluble phloem proteins which are present in lower phloem-specific enzymes. By promoter-reporter gene
abundance in the phloem sap are still being identified fusions, Ishiwatari and coworkers were able to show that
(Marentes and Grusak, 1998). The term ‘sieve tube the thioredoxin promoter drives reporter gene mRNA
exudate proteins’ (STEPs) has been introduced to describe accumulation in SEs, whereas activity of the
the diversity of unknown phloem proteins (Sakuth et al., b-glucuronidase (GUS ) reporter protein was not detected
1993). The soluble proteins could be involved in sieve in SEs, but in the neighbouring CCs ( Sasaki et al., 1998).
tube plugging, SE maintenance or information transfer The phloem sap not only contains proteins, but also
between sink and source organs (Raven, 1991). Ubiquitin dierent mRNA species. Besides thioredoxin mRNA,
and chaperones have been detected in the phloem sap of several other mRNAs have been detected in the phloem
Ricinus (Schobert et al., 1995) and the polyubiquitin sap of rice by RT-PCR, e.g. actin mRNA, whereas
cDNA was found in a cDNA library from the phloem of detection of cystatin mRNA failed (Sasaki et al., 1998).
pine (Alosi-Carter et al., 1995). Ubiquitin and chaperones Fisher et al. (1992 ) collected phloem proteins of wheat
may be involved in the degradation of phloem proteins using radiofrequency microcautery and analysed the turn-
in enucleate SEs, explaining the high selectivity of the over of about 100 soluble phloem proteins by 35S-
autolytic degradation processes occurring during SE methionine labelling. From a total of almost 200 soluble
maturation. proteins, they were able to separate about 100 polypep-
tides by two-dimensional gel electrophoresis, which turned
Stability and turnover of SE proteins over rapidly. The analysis of rice phloem sap by SDS-
PAGE indicated that it contains over 150 dierent pro-Although it has been known for many years that enucleate
SEs contain soluble proteins at low concentration, it is teins ( Nakamura et al., 1993 ). After application of (c-
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938 Ku
¨hn et al.
32P)ATP to leaf blades, the authors observed a light- (Mu
¨nch, 1930). The model of mass flow-driven sucrose
translocation would require the osmotic isolation of thedependent phosphorylation and dephosphorylation of
dierent phloem proteins. Proteins in the rice phloem sap conducting elements of the phloem, namely the SECCC.
According to a more realistic model of Mu
¨nch’s theory,were also phosphorylated in vitro. It has been hypothes-
ized that phosphorylation of phloem proteins is part of a sucrose is lost by diusion during transport towards the
sink organs, requiring an active retrieval mechanism alongsignal-transduction system.
The presence of ubiquitin and chaperones in the phloem the translocation path. Loss of sucrose by diusion
depends strongly on the flow rate: the higher the rate ofsap (Schobert et al., 1995), as well as the presence of
thioredoxin h disulphide-reductase as a potential repair translocation, the lower the loss of sucrose by diusion
into the tissue surrounding the SECCC.enzyme or regulatory enzyme ( Ishiwatari et al., 1995),
are hints that SEs have the capacity to metabolize pro-
teins. Phloem exudate collected from dierent species by
Models of phloem loading
the aphid method was shown to have the ability to
incorporate 32PO3
4into a series of organic compounds Two mechanisms of phloem loading are discussed: (1 )
the symplasmic path via PD, connecting mesophyll cellslike ATP, very similar to those synthesized after the whole
phloem is incubated with 32PO3
4. Thus it was concluded, and the conducting elements of the phloem and (2 ) the
apoplasmic route, in which sucrose is first exported intothat phosphorylating enzymes are present in the phloem
sap which had been deprived of mitochondria by centrifu- the apoplast and is then taken up into the SECCC by an
energy-dependent transport system. Plants appear to showgation (Becker et al., 1971). Neumann and Wollgiehn
found as early as 1964, that mature SEs of Vicia faba are preferences for dierent types of transport molecules
based on their mode of phloem loading (van Bel andable to incorporate 3H-phenylalanine (Neumann and
Wollgiehn, 1964). Thus it seems possible that phloem Gamalei, 1992).
Anatomical features have been used to categorize plantproteins are not only turned over in CCs, but to a certain
extent in mature SEs as well. species based on their mechanism of phloem loading
(Gamalei, 1989; van Bel, 1993; Flora and Madore, 1996).The mobility of phloem proteins was confirmed by
interspecific and intergeneric graft experiments (Golecki According to the plasmodesmal frequency between the
SECCC and the surrounding cells of minor veins, whereet al., 1998 ). Sieve pores do not hinder the phloem
translocation of proteins, and soluble phloem proteins phloem loading is thought to take place, plants can be
classified into three groups: open type I minor veins haveare not anchored to the SE plasma membrane.
Interestingly, stock-specific proteins have been detected multiple PD between the SECCC and phloem paren-
chyma, bundle sheath or mesophyll cells. CCs are oftenby immunolocalization after grafting, not only in the SE
of the scion, but also in the CCs, arguing for the capacity modified into intermediary cells, and phloem loading is
thought to occur symplasmically. The best example of aof macromolecules such as proteins to trac through the
Pore-Plasmodesmata-U nits connecting SEs with CCs type I minor vein configuration are cucurbits (e.g.
Cucurbita melo with extensive IC configuration). In these(PPUs, see below) in both directions. Thus it seems that
macromolecular tracking via PPUs is bi-directional. species, galactosyl oligosaccharides such as stachyose,
ranose and verbascose rather than sucrose are the
principal transport sugars (Gamalei, 1985; Bachmann et
Sucrose as the main transport molecule
al., 1994). In the latter case the osmotic potential per
loaded carbon atom is lower than that of sucrose, allowingMost plants use sucrose as the transport metabolite for
long distances. Physicochemical properties of the sucrose only reduced translocation rates. This results in a higher
energy loss due to retrieval of diused photoassimilates,molecule could be one reason for this preference. Even
in solutions of high concentration, such as phloem sap, but at the same time requires less energy for phloem
loading per carbon atom, thus potentially leading to awith a sucrose concentration in the range of 200 to
1600 mM, viscosity is relatively low allowing high translo- more or less comparable energy balance. A physiological
hint for symplasmic carrier-independent phloem loadingcation rates (0.5 to 3 m h1). An additional advantage
of sucrose over reducing sugars is the chemical stability is the insensitivity toward thiol group-modifying agents
such as p-chloromercuribenzene-sulphonic acid (PCMBS)of the non-reducing disaccharide. Furthermore, sucrose
creates a high osmotic potential per carbon atom, in the or N-ethylmaleimide ( NEM ) (Giaquinta, 1976; Delrot et
al., 1980; van Bel et al., 1994 ).phloem sap, a key parameter for translocation eciencies
within long tubes (van Bel, 1996). In minor veins classified into the primitive closed type
IIa group (e.g. Nicotiana tabacum), symplasmic connectiv-The translocation rates in the phloem are thought to
be driven by mass flow, due to water molecules entering ity of the SECCC is low to moderate, whereas in type IIb
(e.g. Vicia faba, Fabaceae) the SECCC is almost sym-the phloem with sucrose loading and thereby creating an
osmotic pressure gradient along the transport route plasmically isolated from the surrounding cells, making
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Sucrose transport in higher plants 939
it indispensable to include an apoplasmic step in the can be inhibited by PCMBS treatment suggesting that
membrane proteins are involved in an active transportprocess of phloem loading. In a number of ‘apoplasmic’
species, prominent transport carbohydrates are sugar process (Delrot et al., 1980).
Species belonging to the intermediate type I-IIa groupalcohols like mannitol and sorbitol ( Loescher, 1987).
These plants often show a modification of either the CCs are considered to be symplasmic and apoplasmic loaders
at the same time (e.g. Catharanthus roseus, which hasor the parenchyma cells of minor veins, to transfer cells.
However, the significance of the classification has been both CCs and ICs in the minor vein phloem and which
translocates sucrose as well as ranose within thecriticized (Turgeon, 1996). A number of type I plants,
especially trees, were shown mainly to transport sucrose. phloem).
The Solanaceae family represents a polytypic family.Furthermore, the bias towards having monotypic families
is due to the type of analysis, in most cases only a single Datura belongs to the intermediate type I-IIa among the
Solanaceae (1–10 PD mm2interface), whereas Nicotianaspecies was analysed per family. Thus, a generalization
of the correlation between structure and the loading tabacum and Solanum tuberosum belong to the primitive
closed type IIa (with 0.08 PD mm2and 0.12 PD mm2mechanism requires further analysis, i.e. antisense repres-
sion of sucrose transporters in a variety of species interface, respectively) (Gamalei, 1991).
Physiological and anatomical classification of uptakebelonging to dierent structural categories.
Transfer cells are characterized by numerous cell wall do not always agree. Recently, it was published that
sucrose uptake in peach can be abolished by PCMBSinvaginations, allowing up to a 20-fold increase in the
plasma membrane surface area (Pate and Gunning, 1972; treatment indicating an apoplasmic uptake system.
However, based on anatomical criteria, the transportGunning, 1977; Bonnemain et al., 1991). The presence of
transfer cells in specific areas seems to correlate with the system had been considered to be a symplasmic one.
These findings make it imperative to re-evaluate theexistence of intensive solute fluxes between symplasm and
apoplasm (Gunning, 1977; Oer et al., 1989 ). Transfer methods used for classification.
Both classification by anatomical characteristics as wellcells exist either as modified CCs (type A) with high
cytoplasmic density and cell wall invaginations all over as inhibitor studies using PCMBS have been discussed
controversially. Classification according to simple numer-the cell, or as modified phloem parenchyma cells (type
B), with polarized invaginations that are highest at the ical data of plasmodesmal frequency implies that PD are
holes allowing the diusion of various substances includ-opposite side to the SECCC. It has been shown that the
transfer cell wall is pectin-rich and can be highly hydrated, ing macromolecules. Recent studies revealed that PD are
dynamic, complex structures, through which the transporta phenomenon which facilitates solute uptake from the
apoplasmic space. Appearance of transfer cell modifica- of macromolecules is highly regulated (Lucas et al., 1993;
van Bel and Oparka, 1995). Gamalei and coworkerstion in minor veins correlates with the sink–source trans-
ition of young leaves, an indication that the increase in found that temperature-induced ER rearrangement cor-
relates with an inhibition of photosynthate transport andthe plasma membrane surface area is important for
apoplasmic phloem-loading processes ( Turgeon, 1996 ). starch accumulation in plants with a symplasmic minor
vein configuration (Gamalei et al., 1994). Another interes-Embryonal transfer cells of Vicia faba show a polarized
organization, with the localization of wall ingrowths at ting finding which indicates how important PD are for
the mode of phloem loading, is demonstrated in a maizeone pole of the cell and PD at the other pole (Oer et
al., 1989; Bonnemain et al., 1991). This polarized organ- mutant with an abnormal accumulation of starch and a
lack of phloem export, both of which indicate a disturb-ization is accompanied by an asymmetric distribution of
the plasma membrane bound H+-ATPase (Bouche
´-Pillon ance in phloem loading. Russin and co-workers could
show ultrastructurally, that the PD between bundle-et al., 1994 ). Proton pumps are more numerous in the
area of plasma membrane infoldings where active nutrient sheath and phloem parenchyma cells were covered by
wall material, and that the path thus was discontinuous.uptake is assumed to take place ( Harrington et al.,
1997a). ATP hydrolysis by an ATP-energized proton Moreover, the suberin lamellae in the outer and radial
walls of the bundle sheath cells forms a barrier foreux pump provides a gradient for inward proton
coupled substrate transport across the loading membrane apoplasmic solutes ( Russin et al., 1996).
On the other hand, inhibitor studies or fluorescent dyeof phloem cells (Baker, 1978 ). It is thus likely that in
closed type IIb plant species, the main solute fluxes occur microinjection for determining the plasmodesmal SEL
are indirect or invasive techniques, and it can not beat the membranes of transfer cells.
The best example of plants showing a type IIb minor excluded that observed phenomena are due to secondary
eects of the treatment.vein configuration with the development of transfer cell
anatomy, are species belonging to the Faboidae family. In symplasmic models, sucrose transport through PD
has to overcome a concentration gradient. It is still notSucrose is the major transport form of photoassimilates
in these plant species. Sucrose uptake into the SECCC fully understood how the sieve tube system from sym-
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940 Ku
¨hn et al.
plasmic phloem loaders can increase its already high to a specific physiological task. One example is the PD
of CCs, modified into intermediary cells, which can beosmotic potential by additional solute uptake. Sucrose/
proton cotransport is supposed to be unnecessary, as found in many cucurbits and in several other families;
another are the PD between SEs and CCs. These twosucrose does not have to cross membranes; storage in the
vacuole or regulation at the tonoplast membrane is not special cases of asymmetrically branched PD will now be
described.possible since SEs do not have vacuoles. Nevertheless the
existence of a symplasmic connection between mesophyll
cells and minor veins has been demonstrated, e.g. by the Pore-plasmodesma units (PPUs)
dye tracer method in the symplasmic loader Ipomoea These plasmodesmal structures are branched at their CC
tricolor (Madore et al., 1986). Solutions for the energy end and single where they join the SE and frequently
problem in symplasmic loaders have been proposed by contain a large central cavity. As the nature of these PD
several authors (Turgeon and Beebe, 1991; Gamalei et diers to most, having a pore-like orifice at their SE end,
al., 1994). they have been designated pore-plasmodesma units
Turgeon proposed the polymerization trap hypothesis (PPUs) (van Bel, 1996). They dier from PD between
of symplasmic loaders (see below) (Turgeon, 1991). An parenchyma cells in organization and diameter and appar-
alternative theory for the mechanism of symplasmic ently also in regulation. Whereas the SEL of PD is about
phloem loading has been proposed by Gamalei (Gamalei, 800–1000 Da, which is similar to that reported for animal
1985; Gamalei et al., 1994). In intermediary cells of gap junctions (Spray and Bennett, 1985), SELs of PPUs
symplasmic species, a vesicular labyrinth seems to change seem to have much higher limits. The SEL of PPUs in
in volume during the transit of photosynthate. Thus, it the fascicular phloem of Vicia faba has been determined
could be possible to keep the concentration gradient low using fluorescent dextrans to be at least 10 kDa ( Kempers
between the cytoplasm of mesophyll cells and phloem and van Bel, 1997).
cells just by subcellular compartmentation of the trans- PPUs seem to be permanently open. In other tissues,
ported sugars. The energy needed for the accumulation closure of PD in response to wounding by microinjection
of sugars in this vacuolar-vesicular endoplasm is compar- has been observed, while PPUs seemed to be unable to
able to that invested for apoplasmic loading (van Bel, close ( Kempers and van Bel, 1997 ). It would be interes-
1996). For an extensive review on the role of PD in ting to investigate whether viral movement proteins,
symplasmic phloem loading, see Turgeon (1996). which spread specifically via the phloem, are able to
modify the molecular exclusion limit of these structures.
The role of plasmodesmata in phloem loading
Intermediary cell plasmodesmata
One problem in studying the role of PD is that they
constitute extremely small functional units and the reso- Minor vein CCs modified into intermediary cells ( IC )
can be found in plant species that are mostly describedlution of electron microscopes is limited. Electron micro-
graphs are only static images of fixed cells, whereas PD as symplasmic phloem loaders (Cucurbitaceae). IC are
connected with surrounding cells, namely the phloemappear to be dynamic and highly regulated devices, which
are able to open and close according to the environmental parenchyma or bundle sheath via numerous asymmetric-
ally branched PD. The neck region of these special PDconditions ( Kragler et al., 1998).
Methods like High Resolution S canning E lectron (having more branches at their IC end) is narrower at
the IC end, allowing them to act as a size discriminationMicroscopy (HRSEM ), Confocal Laser Scanning
M icroscopy (CLSM ), microinjection of fluorescent dyes, filter. The polymerization trap model ( Turgeon and
Gowan, 1990), which illustrates one possible mechanismGFP-tagged virus movement, biolistic bombardment or
immunolocalization of plasmodesmal components could of symplasmic phloem loading, is based on a selective
filtration by these narrowed sphincter regions of understand plasmodesmal functioning. Recently, the
association of cytoskeletal elements like actin or myosin- Thus the size discrimination function of PD inhibits
diusion back into the bundle sheath/mesophyll area oflike proteins with plasmodesmal structures was shown in
higher plants and in Chara corallina ( White et al., 1994; ranose and stachyose, both of which are larger sugar
molecules, synthesized from sucrose after symplasmicBlackman and Overall, 1998; Radford and White, 1998).
It is hypothesized that these cytoskeletal elements could loading into the IC. Enzymes involved in ranose or
stachyose synthesis have been immunolocalized to inter-play a role in the targeting and transport of macro-
molecules through PD. They could also play a role in mediary cells (Holthaus and Schmitz, 1991; Beebe and
Turgeon, 1992). The galactosyl-oligosaccharides are sub-cell-to-cell transport by interaction with the ER tubule
within PD, either by rearranging it, or by moving it sequently loaded (again via PD) into the SE. To maintain
a low sucrose concentration in the cytoplasm of IC andthrough the PD.
In some cases the structure of PD is closely connected a sucrose concentration gradient between bundle sheath
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Sucrose transport in higher plants 941
and IC, sucrose is removed from the cytoplasm by the phloem, an apoplasmic invertase could be involved
in sucrose degradation and the sucrose cleavage prod-subcellular compartmentation or by storage in the vacu-
ole. Ultrastructural analysis of the IC revealed that they ucts could be imported via monosaccharide trans-
porters (Turgeon, 1989; Wright and Oparka, 1989).are highly vacuolated compared to normal CCs.
Monosaccharide transporters which are specifically
expressed in sink tissues have been isolated from
Arabidopsis and tobacco (Sauer et al., 1990; Sauer and
Phloem unloading
Stolz, 1994). However, active monosaccharide transport
The mechanisms of phloem unloading are less well charac- has also been detected in plasma membrane vesicles from
terized than the mechanisms of phloem loading. It is sugar beet leaves ( Tubbe and Buckhout, 1992 ). The
assumed that the path of unloading can not only vary function of these transporters might be to prevent accu-
between species, but also in a tissue-dependent or even mulation of hexoses in the apoplast. Furthermore, under
developmentally regulated manner (Turgeon, 1989). The certain conditions, source tissues can switch to sink
coexistence of several unloading mechanisms in one function. Typical examples are responses to wounding or
organism (or in one sink organ) was discussed. In tomato pathogen infection, where dramatic reallocation of nutri-
fruit, for example, it is possible that there is a develop- ents is required to sustain defence and repair responses.
mental switch from symplasmic to apoplasmic phloem In agreement with this, the expression of a monosacchar-
unloading (Miron and Schaer, 1991; Oer and Horder, ide transporter is up-regulated locally in leaves after
1992; Ruan and Pattrick, 1995). wounding by pathogen infection (Truernit et al., 1996).
The eciency of phloem unloading depends, among
other factors, on the sink strength of the corresponding Seeds
tissue. The sink strength is determined by the activity of
enzymes involved in sucrose catabolism and/or starch Phloem unloading in seeds is well characterized. In most
species, the embryonal tissue is symplasmically isolatedsynthesis.
Three dierent models of phloem unloading are discus- from the maternal tissue, a strong indication for apo-
plasmic solute transport into the seeds ( Thorne, 1985).sed. In sink organs, the assimilate concentration is higher
in the phloem than in the surrounding sink tissue, thus Detailed studies regarding the role of osmotic pressure in
the regulation of phloem unloading in seeds were per-unloading by facilitated diusion along a concentration
gradient would be one possibility. In order to maintain a formed by Patrick (1993). A detailed description of the
pathway and mechanism cannot be covered in this review.constant concentration gradient, sucrose is either con-
verted into osmotically less ecient storage molecules Furthermore, several excellent reviews have been pub-
lished recently on this topic (Oer et al., 1989; Patrick,such as starch or amino acids, or is cleaved or stored in
subcellular compartments, e.g. in the vacuole. 1997; Weber et al., 1998).
TubersSink leaves
Symplasmic phloem unloading via PD has been postu- The symplasmic connections between the conducting
phloem cells and the storage parenchyma cells of potatolated for sink leaves of sugar beet (Schmalstig and Geiger,
1985, 1987) and tobacco (Turgeon, 1987), as unloading tubers are numerous, thus indicating that symplasmic
phloem unloading is possible (courtesy of A van Bel,cannot be inhibited by thiol-group modifying agents
or anoxia. personal communication, depicted in Frommer and
Sonnewald, 1995). Investigations with fluorescent dyesUsing non-invasive imaging techniques, Roberts and
coworkers compared phloem unloading of the membrane- indicate that metabolites can also be loaded from the
apoplasmic space into the parenchyma cell cytosol byimpermeant, fluorescent solute carboxyfluorescein (CF )
with that of a green fluorescent protein (GFP)-tagged endocytosis (Oparka and Prior, 1988 ). Nevertheless, suc-
rose transport activities, which can be inhibited bypotato virus X in sink leaves of Nicotiana benthamiana.
Unloading of both solute and virus occurred predomi- application of PCMBS have been detected in potato
tubers ( Wright and Oparka, 1989). As mentioned above,nantly from the class III vein network, a highly branched
veinal system found between class II veins. The minor sucrose transporter proteins from higher plants are sensit-
ive towards thiol-group modifying agents such as PCMBSveins (classes IV and V) played no role in solute or virus
import. During the sink–source transition, phloem (Giaquinta, 1976; Delrot et al., 1980 ). Expression of a
yeast invertase in the apoplast of potato tubers led to aunloading of CF was inhibited from class III veins before
the cessation of phloem import through them, suggesting reduced number of tubers, but simultaneously to an
increase in tuber yield (Heineke et al., 1992), indicatinga symplasmic isolation of the phloem in class III veins
before its involvement in export (Roberts et al., 1997 ). that during unloading sucrose is at least partially released
into the apoplasmic space. It cannot be excluded that soAfter apoplasmic, carrier-mediated sucrose release from
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942 Ku
¨hn et al.
far unknown sucrose carriers or mechanisms, are involved
Structure of sucrose transporters
in this process (Oparka et al., 1992 ). Amino acid sequence comparison of the two sucrose
transporters StSUT1 and SoSUT1 which were isolated
Roots by yeast complementation, show 83% similarity and 68%
identity (Riesmeier et al., 1993 ). Their predicted molecu-Several studies regarding phloem unloading in roots came
to the same observation, that unloading in this tissue lar mass is about 54 kDa, while hydrophobicity analysis
indicates a structure for an integral membrane proteinoccurs symplasmically ( Dick and ap Rees, 1975; Farrar,
1985, 1992; Warmbrodt, 1987). with 12 putative transmembrane domains, with a central
hydrophilic loop, resulting in the characteristic 6-loop-6-Unloading of a fluorescent dye in Arabidopsis roots
was visualized using confocal laser scanning microscopy structure described for several cation/substrate-
cotransporters ( Henderson, 1990; Kaback, 1992). This is(CLSM) to occur only at the root tip, from SEs of the
protophloem. Unloading of the fluorescent dye could not the characteristic feature of the family of the so-called
major facilitator superfamily (MFS) (Marger and Saier,be observed in other regions of the root, where SEs of
the metaphloem are active (Oparka et al., 1994 ). 1993). The second loop of the transporters contains a
highly conserved motif ( RXGRR), which can be foundThese results are consistent with the frequency of PD
in both regions of the root: the SECCC of the so-called at the same position in the E.coli lactose permease
(Henderson, 1990 ). Other structural motifs have beentransport phloem (metaphloem) is symplasmically almost
isolated from the surrounding tissue (van Bel, 1993 ). It described in detail ( Ward et al., 1998 ).
can not be excluded, that in this region of the phloem,
carrier-mediated apoplasmic sucrose unloading takes
place. Schulz (1994) found in pea cotyledons that
Biochemistry of SUT1-mediated sucrose uptake
unloading of 14C-labelled sucrose occurs mainly along Sucrose transport characteristics of StSUT1 and SoSUT1
the elongation zone at the root tip, a region which were first determined in yeast cells by 14C-labelled sucrose
exclusively contains mature SEs of the protophloem. uptake studies. Sucrose uptake was pH-dependent, with
These data suggest that symplasmic unloading from cells aKmof 1.5 mM and 1 mM sucrose for SoSUT1 and
of the protophloem is the most important pathway of StSUT1, respectively. Uptake can be inhibited by maltose
assimilate unloading in the root. (Kmof 5 mM for SoSUT1 and 10 mM for StSUT1 ), by
protonophores such as 2,4-dinitrophenol (2,4-DNP) and
carbonyl-cyanid-m-chlorophenylhydrazone (CCCP), and
Characterization of sucrose transporters
by agents covalently modifying specific amino acid res-
idues such as PCMBS and diethylpyrocarbonate ( DEPC ).Complementation of yeast is a powerful tool to isolate
plant membrane proteins (Frommer and Ninnemann, The eect of PCMBS can be partially reversed by the
addition of dithiothreitol (DTT) (Riesmeier et al., 1992,1995). At first appearance, Saccharomyces cerevisiae is
unsuitable for isolating plant sucrose transporters by 1993 ). These data were in accordance with previous
measurements using leaf discs and plasma membranecomplementation due to the excellent growth of baker’s
yeast on sucrose-containing media. This is due to the vesicles (Giaquinta, 1976; Delrot et al., 1980) and were
confirmed by Lemoine et al. (1996) with potato plasmasecretion of an invertase which cleaves sucrose extracellu-
larly. Thus, wild-type yeast was modified by deleting the membrane vesicles (for reviews see Delrot, 1989; and
Bush, 1993).secreted invertase activity and simultaneously inserting
the potato sucrose synthase gene, thereby enabling yeast The transport mechanism of StSUT1 was measured
electrophysiologically in Xenopus oocytes using thecells to metabolize ingested sucrose. Using this comple-
mentation system, it was possible to isolate cDNAs of 2-electrode voltage clamp and radiotracer flux method.
SUT1 mediated sucrose-dependent inward currents andtwo plant sucrose transporters from spinach and potato
(Riesmeier et al., 1992, 1993). The yeast complementation displayed Michaelis-Menten-type kinetics with a 1:1
H+/sucrose stoichiometry (Boorer et al., 1996). Thesystem also allowed the biochemical characterization of
SUT-mediated sucrose uptake (see below). stoichiometry is in accordance with measurements using
plasma membrane vesicles from sugar beet (Bush, 1990;Heterologous screening of cDNA or genomic libraries
was successfully used to identify homologous genes from Slone et al., 1991 ) or electrophysiological measurements
with AtSUC1 from Arabidopsis (Zhou et al., 1997). Theother species, as well as to show the presence of more
than one carrier within a single species. Detailed sequence anities of SUT1 for H+and sucrose were voltage-
dependent. A model can be deduced from the electro-comparisons and phylogenetic studies show that, in most
cases, gene amplifications must be relatively old, thus physiological data, in which protons bind to StSUT1
before sucrose and then both ligands are transportedpredicting specific functions for the individual members
of the family (Rentsch et al., 1998). simultaneously across the membrane.
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Sucrose transport in higher plants 943
Physiological role of SUT1 in Solanaceae
ultrastructurally (Schulz et al., 1998 ). In antisense plants,
starch grains were prominent throughout the mesophyll
Inhibition of SUT1 expression in potato and tobacco and bundle sheath cells, but decreased in number and
(Riesmeier et al., 1994; Ku
¨hn et al., 1996; Bu
¨rkle et al., size in phloem parenchyma and CCs. Also oleosomes
1998) using the antisense approach led to comparable were found in the mesophyll and vascular parenchyma
defects in transgenic plants. The antisense plants showed cells, indicative of a universal build-up of assimilates in
increased concentrations of soluble sugars and starch in the leaf at the border of the SECCC.
their source leaves, as expected for a block in the transport The observed eects of the CC-specific inhibition, qual-
of sucrose into the phloem cells. Sucrose and starch were itatively evoking the identical distribution of starch as
increased 5–10-fold and hexoses even more. Phos- the constitutive SUT1-inhibition, emphasize that the loca-
phorylated hexoses and other intermediate metabolites of tion of the bottleneck of phloem loading is at the SECC
the sucrose biosynthetic pathway were unchanged in these boundary. It can not be excluded that pre-phloem loading
plants compared to wild-type plants. The leaves developed occurs by symplasmic transfer of assimilates from the
a characteristic phenotype which could be related to a mesophyll up to the SECC boundary, but thereafter
change in osmotic conditions within the leaf tissue due assimilates are released into the apoplast, and are sub-
to the accumulation of carbohydrates. Development of sequently loaded into the SECCC. Taken together, the
this phenotype strongly depends on the light period and eects observed in antisense plants demonstrate that
intensity ( Ku
¨hn et al., 1996). A similar accumulation of SUT1 is essential for sucrose loading into the phloem via
soluble carbohydrates was found when phloem transport an apoplasmic route and also possibly in inter-mesophyll
was inhibited by treatments like heat- or cold-girdling transport as well.
(Grusak et al., 1990; Krapp et al., 1993). The leaves of An alternative way to check the physiological role of
tobacco antisense plants showed an impaired ability to sucrose transporters could be the analysis of knock-out
export recently fixed 14CO2and were unable to export mutants which can be identified, for example, by screening
transient starch during extended periods of darkness, T-DNA tagged Arabidopsis mutants by PCR ( Krysan et
leading to an accumulation of soluble carbohydrates and al., 1996). So far, no successful identification of sucrose
a reduction in photosynthetic activity. Autoradiographs transporter mutants has been described.
of leaves showed a heterogenous pattern of CO2fixation
which is maintained even after 24 h of darkness, sug-
Localization of SUT1 in the phloem
gesting that movement of photosynthate between meso-
phyll cells may also be impaired ( Bu
¨rkle et al., 1998). Tissue-specific Northern blot analysis revealed the main
Transgenic plants with a reduced amount of sucrose expression of SUT1 in mature leaves and, to a lower
carrier mRNA showed a reduction in the development of extent, in sink organs. GUS reporter gene expression
the root system and delayed or impaired flowering, and under the control of the LeSUT1 promoter drives expres-
in potato antisense plants, tuber yield was dramatically sion in potato plants in the phloem (Hirner, unpublished
reduced. results). The SUT1 expression pattern was comparable
Antisense inhibition of SUT1 expression using the to the analogous transporter AtSUC2 from Arabidopsis
CC-specific rolC promoter led to similar eects in trans- thaliana (Truernit and Sauer, 1995). GUS staining was
genic potato plants ( Ku
¨hn et al., 1996). It is interesting also found in the transport and delivery phloem such as
that sucrose uptake in plasma membrane vesicles isolated the vasculature of potato tubers, suggesting a role of
from constitutively inhibited potato antisense plants was SUT1 not only in phloem loading, but also in retrieval
shown to be significantly reduced, whereas sucrose uptake during long-distance transport and in phloem unloading
by vesicles isolated from phloem-specifically inhibited in sink organs. These expression data have been confirmed
antisense plants was not reduced compared to wild-type by RNA in situ hybridization ( Riesmeier et al., 1993).
plants (Lemoine et al., 1996). The uptake measurements To dissect out the role of the transporter in dierent
correlate with Western blot data, in which the amount of plant organs, tissue-specific promoters have been used to
SUT1 protein was not reduced in rolC plants, whereas decrease SUT1 expression either in the phloem (rolC
almost no SUT1 protein could be detected in constitutive promoter) or in potato tubers (B33 patatin promoter).
antisense plants. The authors concluded that SUT1 may Several independent transformant lines showed reduced
also be expressed to low levels in cells other than phloem SUT1 mRNA levels if antisense expression was under the
cells, masking the actual amount of sucrose transport and control of either the rolC or the patatin promoter ( Ku
SUT1 protein in the phloem. So far, it was not possible et al., 1996). The rolC promoter has been shown to drive
to define the control coecient of SUT1, which would CC-specific expression in transgenic tobacco plants
indicate the significance of the transporter in the process (Ku
¨hn et al., 1996). Ecient antisense inhibition of SUT1
of phloem loading. using this CC-specific promoter, together with the pheno-
typical symptoms of these antisense plants, indicates thatBoth sets of potato antisense plants have been analysed
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944 Ku
¨hn et al.
SUT1 mRNA transcription takes place in CCs. To deter- re-evaluate the paradigm of the absence of ribosomes in
mature SEs, using immunolabelling techniques formine expression at the cellular level, immunolocalization
studies were performed. Immunolocalization using example.
Interestingly, SUT1-protein is also present in young,immunofluorescence and silver-enhanced immunogold-
staining, detected SUT1 in plasma membranes of enuc- still nucleated SEs found in elongating internodes ( Ku
et al., 1997 ) ( Fig. 1) . This finding fits nicely with olderleate SEs of tobacco, potato and tomato ( Ku
¨hn et al.,
1997). SUT1 was found both in adaxial ( inner) and reports that these cells may already be active in sugar
transport (Schumacher, 1933; Lackney and Sjo
¨lund,abaxial (external ) phloem in leaves and stems, respect-
ively. The protein was not only found in minor veins of 1991 ). It would be interesting to answer the question of
the half-life of the transporter. Prolonged dark treatmentsource leaves but also in stems, petioles and roots.
Interestingly, SUT1 could only be detected in the SE of of potato plants led to a rapid decrease in StSUT1 protein
and investigations using cycloheximide as a translationalthe root metaphloem and not in the protophloem SE,
which are not accompanied by CCs, but which are highly inhibitor indicate a high turnover rate, i.e. within a few
hours. A more detailed determination of the half-life willconnected by PD to their surrounding cells. This is in
agreement with the finding that unloading from pro- help to elucidate at which stage of development SEs start
to be dependent upon protein or RNA imported fromtophloem SEs is thought to occur symplasmically (Oparka
et al., 1994). In situ hybridization at the EM level also CCs.
showed that SUT1 mRNA localizes to both SEs and
CCs, and is preferentially associated with the orifices of
the PD. The mRNA localization, together with the
CC-specific antisense inhibition of SUT1, indicate that By heterologous screening of cDNA or genomic libraries
using the transporters isolated by yeast complementationtranscription of SUT1 mRNA occurs in CCs. A con-
sequence of the dierential distribution of SUT1 mRNA as probes, it was possible to identify several other sucrose
transporter genes from a variety of plants (Fig. 2). Asand protein is that either the mRNA or the protein has
to be translocated via the specific pore plasmodesma units for monosaccharide transporters, sucrose transporters
also seem to belong to a large gene family. Several sucrose(PPU) from the CC into the SE, and that phloem loading
with sucrose occurs directly at the plasma membrane of transporters are known from Lycopersicon esculentum
(LeSUT1,LeSUT2 and LeSUT4: L Barker, unpublishedSEs. Tracking of macromolecules through PD is a well-
known phenomenon for viral cell-to-cell movement in results), from Nicotiana tabacum (NtSUT1:(Bu
¨rkle et al.,
1998); NtSUT3: R Lemoine, unpublished results), Betaplants, which is usually facilitated by the interaction of
viral movement proteins with plasmodesmal components vulgaris (BvSUT1: accession number 1076257 ) and
Plantago major (PmSUC1 and PmSUC2: Gahrtz et al.,(Lucas and Gilbertson, 1994; Almon et al., 1997). It has
also been described for endogenous plant proteins, e.g. 1994 ). The search for new sucrose transporters from
Arabidopsis thaliana is facilitated by the Arabidopsisthe transcription factor KNOTTED1 ( Kn1 ) ( Lucas et
al., 1995). In several cases, both protein and mRNA are genome project: so far at least six dierent sucrose
transported from cell to cell. The mRNA molecules could
be guided to the PPUs by the help of cytoskeletal compon-
ents, whereas the SUT1 protein could be targeted to the
SE either as part of the ER or as part of the plasma
membrane, both of which are continuous through the PD
(Overall and Blackman, 1996 ).
Asymmetric mRNA distribution and polarized trans-
port of mRNA within and between cells has been observed
for example during Drosophila oogenesis (St Johnston,
1995). If SUT1 mRNA is translocated, then it has to be
translated in the SE, which lose the majority of their
ribosomes during maturation. In 1964, Neumann and
Wollgiehn studied the incorporation of 3H-uridine and
3H-phenylalanine into young nucleated and mature enuc-
leated SEs from Vicia faba. They could detect 3H-uridine
incorporation into RNA molecules only in the early
Fig. 1. Immunolocalization of StSUT1 in young still nucleated SEs
stages of SE dierentiation but, in contrast, could detect
right after asymmetric cell division from the neighbouring CC. Nuclei
3H-phenylalanine incorporation into proteins even after
are stained with DAPI. Even at this early stage of SE dierentiation,
the disintegration of the SE nucleus (Neumann and
SUT1 is already detectable in the plasma membrane of SEs, which at
this stage are still autonomous.
Wollgiehn, 1964). It will therefore be essential to
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Sucrose transport in higher plants 945
isolated seeds (Harrington et al., 1997b; Weber et al.,
1997; Tegeder et al., 1999).
The carrot transporters DcSUT1 and DcSUT2 are
dierentially expressed: DcSUT1 was only found in the
green parts of the plant with highest expression levels in
the lamina of source leaves; it was diurnally regulated,
indicating a role in phloem loading. In contrast, DcSUT2
expression was mainly found in sink organs and had no
diurnal expression pattern. This carrot transporter may
be involved in the loading of sucrose into storage paren-
chyma cells (Shakya and Sturm, 1998).
In Plantago and Arabidopsis, immunofluorescence with
specific antibodies detected the SUT1 homologue termed
SUC2, in CCs (Stadler et al., 1995; Stadler and Sauer,
1996). The AtSUC2-promoter drives expression of the
GUS reporter gene in the phloem cells of exporting leaves,
but also in sink organs such as roots, sepals and filaments
of flowers and in young siliques of transgenic Arabidopsis
plants (Truernit and Sauer, 1995). A potential role in
phloem loading and unloading has been discussed as in
the case of SUT1.
Whereas PmSUC2 is mainly expressed in the loading
phloem of source leaf minor veins, PmSUC1, is expressed
in young ovules (Gahrtz et al., 1996 ) and also in leaf
vascular bundles. The cell specificity of PmSUC1 and
Fig. 2. Computer-aided homology analysis by PHYLIP ( Felsenstein,
PmSUC2 was shown immunocytologically: PmSUC2,
1993) of aligned sucrose transporters from tobacco (NtSUT1:Bu
et al., 1998), potato (StSUT1: Riesmeier et al., 1993), spinach (SoSUT1:
which is thought to be responsible for phloem loading
Riesmeier et al., 1992) ; tomato (LeSUT1,LeSUT2,LeSUT4 L. Barker,
was localized to CCs, while PmSUC1 antibodies labelled
unpublished results), Arabidopsis (AtSUC1; AtSUC2: Sauer and Stolz,
starch-containing cells of developing seeds, putatively the
1994); Plantago (PmSUC1; PmSUC2: Gahrtz et al., 1994, 1996) ,
Ricinus (RcSUT1: Weig and Komor, 1996), sugar beet (BvSUT1,
endosperm and the SEs of the transport phloem in petioles
accession number 1076257), rice (OsSUT1: Hirose et al., 1997 ), fava
(Stadler et al., 1998 ).
bean (VfSUT1: Weber et al., 1997), and carrot (DcSUT1,DcSUT2:
These data suggest, that in Plantago, phloem loading
Shakya and Sturm, 1998). The numbers indicate the occurrence of a
branch in 100 bootstrap replicates of a given data set. OsSUT1 was
occurs in CCs, whereas the main retrieval function during
used as the outgroup.
long-distance transport is localized directly at the SE
plasma membrane. Another possibility is stepwise phloem
loading through several dierent cells involving two suc-
rose transporters working in parallel with varying celltransporters have been isolated (AtSUC1 to AtSUC2:
Sauer and Stolz, 1994; AtSUT27: J Ward, unpublished specificities.
It could be possible that dierent sucrose transportersresults; Michel et al., 1998 ). Recently, the cDNAs of two
sucrose transporters from carrot have been characterized are localized in dierent cell types, allowing a division of
labour, or a phloem loading mechanism which occurs in(Daucus carota:DcSUT1 and DcSUT2, Shakya and
Sturm, 1998 ). several steps in several cells. This was first suggested in
1949 by physiological analyses showing a stepwise sucroseAdditionally, SUT1 homologues were found in Ricinus
communis (RcSUT1: Weig and Komor, 1996), Oryza gradient (dierent sucrose concentrations) in dierent
phloem cells (Roeckl, 1949). A stepwise mechanism ofsativum (OsSUT1: Hirose et al., 1997), Vicia faba
(VfSUT1: Weber et al., 1997), Pisum sativum (PsSUT1: phloem loading could involve several sucrose transporters,
which are so far not characterized in detail, nor localizedTegeder et al., 1999 ), Apium graveolens (AgSUT1:
Noiraud et al., 1998), and grape (Vitis vinifera: Picaud et at the cell-specific level in all species.
al., 1998).
Some of these transporters show very specific expres-
Efflux of sucrose into the apoplast
sion patterns. NtSUT3 is expressed exclusively in pollen,
suggesting an important role in pollen loading. In Vicia Characterization of the isolated sucrose transporters has
increased the amount of information about the mechan-seeds, cross-hybridization with sucrose transport genes
could be detected in the transfer cells of cotyledons ism of phloem loading that is now available. However,
what still remains unanswered, is how sucrose exits theresponsible for sucrose loading into the symplasmically
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946 Ku
¨hn et al.
mesophyll cells (its site of synthesis) and enters the responsible for a low-anity sucrose uptake system ( Ripp
et al., 1988; Warmbrodt et al., 1989; Grimes et al., 1992).apoplast. Sucrose eux into the apoplast prior to phloem
loading could occur from the mesophyll cells or directly SBP is a hydrophilic protein, containing an N-terminal
putative membrane spanning domain. The majority ofat the interface of the SECCC. Mechanistically, the release
carrier could be a facilitator or an antiporter. Although the protein is located extracellularly, the N-terminal
domain anchoring it to the membrane (Grimes et al.,a so-called ‘eux’ carrier was biochemically characterized
from the plasma membranes of Beta vulgaris, the corres- 1992; Overvoorde and Grimes, 1994). As the protein has
been localized to the plasma membrane of cells withponding gene has not been isolated (Laloi et al., 1993).
Characteristics obtained argue for the existence of a sucrose uptake capacity, the SEs in spinach and sugar
beet but primarily the CCs in soybean ( Warmbrodt etpassive sucrose eux system that is dierent from the
sucrose/H+symport responsible for phloem loading. The al., 1989 ), one assumes that this protein might be involved
in sucrose transport, while its biochemical propertiesexistence of eux carriers has also been postulated in
unloading processes, where sink cells take up sugars from indicate that it may encode the linear component of
sucrose uptake observed in many transport studies. SBPthe apoplast (Patrick and Oer, 1995; Wang et al., 1995b;
McDonald et al., 1996 ). It is still a matter of debate is able to mediate radiolabelled sucrose uptake on its own
in the SUSY7 mutant (Overvoorde et al., 1996).whether these carriers function as facilitators or as sucrose
proton antiporters. To date, none of these carriers have Interestingly, one of two polypeptides purified by sucrose
anity chromatography from the plasma membranes ofbeen identified at the molecular level ( Patrick and
Oer, 1995). sugar beet leaves, exhibited sucrose transport activity
after reconstitution into proteoliposomes (Li et al., 1994).
As heteromeric structures have been reported for mamma-
Other proteins complementing sucrose uptake
lian amino acid transporters ( Bertran et al., 1992), one
could postulate that SBP may have a regulatory role inComplementation of the artificial yeast mutant SUSY7
with a potato cDNA expression library did not allow the phloem sucrose transport through interaction with SUT1.
Consistent with this, SBP was co-localized withidentification of new sucrose transporter genes other than
the already known SUT1. However, a number of plant sucrose/H+cotransporters in the plasma membrane of
Vicia faba transfer cells in developing seeds, and ingenes have enabled the mutant to grow on sucrose,
including PCP1, that shares significant homologies with cotyledons of Vicia faba ( Harrington et al., 1997a;b). In
developing seeds of Pisum sativum, SBP was localizedzinc finger-containing transcriptional regulators ( Ku
and Frommer, 1995) and, interestingly, a soybean sucrose exclusively in cotlydon epidermal transfer cells, whereas
PsSUT1 and the H+-ATPase were primarily in epidermalbinding protein ( SBP) (Overvoorde et al., 1996). The
phenotype observed in yeast expressing one of these transfer cells, but were also localized to storage paren-
chyma cells (Tegeder et al., 1999) Coexpression of SUT1proteins mimicked the functional complementation by a
sucrose transporter. Therefore, it may be concluded that and SBP in oocytes might be a suitable approach to test
the hypothesis that SBP also has a regulatory function.these proteins suppress the phenotype of SUSY7 by
transcriptional activation of an otherwise inactive endo-
genous sucrose transporter. The presence of such endo-
Stability and regulation of sucrose transporter
genous sucrose transporters has previously been
postulated ( Khan et al., 1973; Santos et al., 1982) which,
however, have never been demonstrated at the molecular The activity of sucrose transporter proteins could be
regulated at dierent levels: at the transcriptional or thelevel. With the completion of the yeast genome sequencing
project and subsequent classification of all of the open post-transcriptional level, which includes mRNA stability,
protein targeting and modification. It is obvious forreading frames corresponding to proteins of the major
facilitator family (Nelissen et al., 1997), characterization sucrose proton symporter proteins that their activity
strongly depends on the proton motive force which isof potential sucrose transporters at the molecular level
has been simplified. That no sucrose transporter has thus established be H+-ATPases. Therefore, the regulation of
sucrose uptake or eux also includes the regulation offar been identified in Saccharomyces cerevisiae,isno
reason to suggest its absence. As no sucrose transport ATPases. Thus, it is important to discriminate between
direct regulatory eects on sucrose transporters and indir-occurs under normal growth conditions, but is only
induced in certain circumstances, the transporter (s) may ect eects via ATPase activity.
be structurally suciently dierent from the known plant
sucrose transporters, that no homology is identifiable.
Transcriptional regulation
The SBP was isolated from soybean cotyledons using
a photoanity labelling technique with a sucrose ana- Transcriptional regulation of the enzymes involved in
sucrose and starch biosynthesis seems to be welllogue and has been identified as a potential candidate
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Sucrose transport in higher plants 947
co-ordinated and depends on the corresponding light unchanged after hormone application (Harms et al.,
1994). Hormone regulation of sucrose uptake has beenconditions, the developmental stage and the nutritional
status. Thus, the expression profile of the sucrose trans- reviewed by Ward et al. (1998).
In contrast to Harms et al. ( 1994) who found that theporter mRNAs from potato and Arabidopsis is under
developmental control and follows the sink-to-source sucrose transporter StSUT1 from potato is not influenced
at the transcriptional level by application of varioustransition ( Riesmeier et al., 1993; Truernit and Sauer,
1995). sucrose concentrations, Chiou and Bush (1998) identified
sucrose as a signalling molecule in a previously unde-The eux of 14C-labelled sugars and the concentration
of soluble sugars and starch in source leaves is diurnally scribed signal-transduction pathway that regulates the
sucrose symporter from sugar beet. Sucrose symporterregulated in potato and tobacco with a maximum rate at
the end of the light period ( Heineke et al., 1994). In activity of sugar beet declined in plasma membrane
vesicles isolated from leaves fed exogenous sucrose viaaccordance with these export data, the sucrose transporter
SUT1 from tomato and potato is diurnally regulated as the xylem transpiration stream, due to a decrease in
symporter message levels, suggesting a drop in transcrip-well ( Ku
¨hn et al., 1997). It can not be excluded that the
light-induced increase of SUT1 transcripts is based on a tional activity or a decrease in mRNA stability. The
decrease in sucrose transporter mRNA levels was revers-circadian regulation of the transporter. However, also
phytohormones such as auxin and cytokinin have been ible and it is discussed that a sucrose-sensing pathway
can modulate transport activity as a function of changingshown to increase the rate of phloem loading (Lepp and
Peel, 1970; Patrick, 1976). Harms et al. (1994) showed sucrose concentrations in the leaf (Chiou and Bush, 1998 ).
This regulation pathway could be an example of ‘sinkthat the transcript levels of the potato sucrose transporter
StSUT1 can be increased after application of phytohor- regulation’, in which decreasing sink demand led to
increased sucrose concentrations in the phloem, down-mones to detached leaves. The eect of auxin (2,4-D)
and cytokinin (BAP) was also detectable on the protein regulating the sucrose symporter. Such a sink regulation
has already been described for photosynthetic geneslevel (C Ku
¨hn, unpublished results). It can be excluded
that the phytohormones act indirectly via promotion of ( Krapp et al., 1993) and invertases (Roitsch et al., 1995).
the H+-ATPase, since the transcript level of the potato
H+-ATPases PHA1 and PHA2 was shown to be Post-transcriptional regulation
One of the most common types of post-translational
regulation of protein activity involves phosphorylation/
dephosphorylation. Very recently, it was shown that
sucrose and valine uptake in leaf discs from Beta vulgaris
can be inhibited by application of okadaic acid (OA ), an
inhibitor of protein phosphatases 1 and 2a (Roblin et al.,
1998). These observations were confirmed by measuring
direct uptake into PMV prepared from leaves infiltrated
with OA to exclude possible secondary eects by OA.
H+-ATPase activity was unaected by OA treatment,
and the amount of transporter proteins did not decrease
as shown by specific antibodies. Roblin and coworkers
therefore suggest that OA acts directly on the activity of
the sucrose and valine transporters by maintaining them
in a phosphorylated form and thus inactivating them. As
mentioned above, phosphorylating enzyme activities
which might be involved in phosphorylation of sucrose
transporters in the SEs have been detected in the phloem
sap (Nakamura et al., 1993).
Targeting of transporters to the plasma membrane
Plant transporters that complement yeast mutants defi-
Fig. 3. Confocal imaging of the localization of GFP-tagged LeSUT1
cient in nutrient uptake must be located in the plasma
expressed in the yeast strain SUSY7 ( Riesmeier et al., 1992). Note that
LeSUT1 is only partially targeted to the plasma membrane of yeast
membrane. In analogy to membrane protein targeting in
cells, while some molecules are retained in some perinuclear structures
yeast, one might assume that it involved import into the
that are probably ER cisternae. The few molecules that reach the
ER membrane and transfer via the Golgi apparatus and
plasma membrane are sucient to complement this sucrose uptake-
deficient yeast strain.
secretory vesicles to a plasma membrane destination. If
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948 Ku
¨hn et al.
Fig. 4. Model explaining the possible translocation pathway of sucrose during pre-phloem loading and sucrose import into the sieve-element-
companion cell complex, which occurs directly at the plasma membrane of SEs as it has been elucidated in solanaceous plants.
the proteins are not recognized correctly by the yeast Nakamura et al., 1993). Thus the main prerequisites for
SE specific turnover and degradation are present.targeting machinery, they may reach their final destination
via unspecific mechanisms (Frommer and Ninnemann,
Secondary effects via regulation of ATPases
1995). Studies examining the expression of tomato SUT1
as a translational fusion with GFP in the SUSY7 mutant, Many secondary active transport processes across mem-
revealed that only a portion of the expressed protein branes depend on the activity of H+-ATPases. Therefore,
reaches its plasma membrane destination (C Ku
¨hn, ATPases are a primary target for regulation. Several
unpublished results) (Fig. 3). A large proportion remains factors can regulate ATPase activity. The H+-pumping
within the cell, forming a strongly labelled perinuclear activity is regulated via an autoinhibitory domain in the
ring. Antibody detection of the SUT1 transporter revealed C-terminal region of the enzyme (Palmgren et al., 1990).
no dierence in its localization whether SUT1 was The fungal toxin fusicoccin strongly stimulates H+-
expressed alone or as a fusion with GFP. Thus, fusing ATPase activity via binding to a protein belonging to the
GFP to the SUT1 protein causes no targeting problems 14-3-3 family, which interacts directly with the autoinhibi-
for the yeast secretory pathway. As the expression of tory domain of the enzyme (Oecking et al., 1994; Jahn et
both of these constructs is able to complement the growth al., 1997). Anaerobiosis probably acts at the level of ATP
of SUSY7 on sucrose, the small fraction of the SUT1 supply for the H+-ATPase (Giaquinta, 1977; Servaites et
protein reaching the plasma membrane is sucient for al., 1979; Thorpe et al., 1979; Maynard and Lucas, 1982).
functional complementation. Salicylic acid (SA) inhibited both valine and sucrose
uptake in sugar beet leaf discs in a concentration-
Turnover dependent manner. SA acts specifically, but does not
aect the amount of sucrose transporter mRNA or pro-Inhibition studies using cycloheximide show that the half-
life of protein turnover is in the range of a few hours tein. It has been discussed whether SA aects sugar and
amino acid uptake via inhibition of the H+-ATPase(Ku
¨hn et al., 1997). The turnover rate of the H+-ATPase
from maize has been shown to be very high, in the range energizing these processes ( Bourbouloux et al., 1998).
of 12 min after auxin treatment ( Hager et al., 1991 ). The
high turnover rate of these proteins indicates that specific
mechanisms are involved possibly requiring ubiquitin-
ation and internalization by endocytosis, as found for Sucrose transporter genes from higher plants are members
of large gene families. In most species, more than oneseveral yeast plasma membrane transport proteins or
mammalian glucose transporters (Hare, 1990; Hein et al., carrier was identified. In ‘apoplasmic’ species, phloem
loading depends on the presence of a secondary active1995). In the phloem sap, ubiquitin was shown to be one
of the major proteins (Schobert et al., 1995 ). The presence sucrose uptake system. Antisense inhibition of the sucrose
transporter SUT1 from the Solanaceae indicated that theof ATP and phosphorylating enzyme activities in the
phloem sap has also been shown ( Becker et al., 1971; carrier is essential for phloem loading. Other members of
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Sucrose transport in higher plants 949
cytoskeleton to plasmodesmata of Chara corallina.The Plant
the sucrose transporter family could be involved in other
Journal 14, 733–741.
functions such as retrieval or phloem unloading.
Bonnemain JL, Bourquin S, Renault S, Oer C, Fisher DG.
Alternatively, phloem loading could occur in a stepwise
1991. Transfer cells: structure and physiology. In: Bonnemain
manner recruiting several dierent transporters, possibly
J, Delrot S, Lucas W, Dainty L, eds. Recent advances in
located in dierent cells. Sucrose transporters are key
phloem transport and assimilate compartmentation. Nantes-
Paris: Ouest Editions, Presses Acade
´miques, 74–83.
proteins in carbon partitioning and are localized at stra-
Boorer KJ, Loo DDF, Frommer WB, Wright EM. 1996.
tegic positions, thus being an important target for regula-
Transport mechanism of the cloned potato H+/sucrose