Plant Cell Physiol. 47(4): pcj011–00 (2006)

doi:10.1093/pcp/pcj011, available online at www.pcp.oupjournals.org

JSPP © 2006

An Important Pool of Sucrose Linked to Starch Biosynthesis is Taken up by Endocytosis in Heterotrophic Cells

Edurne Baroja-Fernandez 1, Ed Etxeberria 2, Francisco José Muñoz 1, María Teresa Morán-Zorzano 1,

Nora Alonso-Casajús 1, Pedro Gonzalez 2 and Javier Pozueta-Romero 1, *

1 Agrobioteknologia Instituta, Nafarroako Unibertsitate Publikoa, Gobierno de Navarra and Consejo Superior de Investigaciones Científicas,

Mutiloako etorbidea zenbaki gabe, 31192 Mutiloabeti, Nafarroa, Spain 2 University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA

;

We have recently shown the occurrence of endocytic

sucrose uptake in heterotrophic cells. Whether this mecha-

nism is involved in the sucrose–starch conversion process

was investigated by comparing the rates of starch accumu-

lation in sycamore cells cultured in the presence or absence

of the endocytic inhibitors wortmannin and 2-(4-morpholy-

nyl-)-8-phenyl-4H-1 benzopyran-4-1 (LY294002). These

analyses revealed a two-phase process involving an initial

120 min wortmannin- and LY294002-insensitive starch

accumulation period, followed by a prolonged phase that

was arrested by the endocytic inhibitors. Both wortmannin

and LY294002 led to a strong reduction of the intracellular

levels of both sucrose and the starch precursor molecule,

ADPglucose. No changes in maximum catalytic activities of

enzymes closely linked to starch and sucrose metabolism

occurred in cells cultured with endocytic inhibitors. In

addition, starch accumulation was unaffected by endocytic

inhibitors when cells were cultured with glucose. These

results provide a first indication that an important pool of

sucrose incorporated into the cell is taken up by endocytosis

prior to its subsequent conversion into starch in hetero-

trophic cells. This conclusion was substantiated further by

experiments showing that sucrose–starch conversion was

strongly prevented by both wortmannin and LY294002 in

both potato tuber discs and developing barley endosperms.

Keywords: Acer pseudoplatanus — Hordeum vulgare — Sink

strength — Solanum tuberosum, Sucrose–starch conversion —

Sucrose synthase.

Abbreviations: LatB, latrunculin B; LY, Lucifer yellow-CH;

LY294002, 2-(4-morpholynyl-)-8-phenyl-4H-1 benzopyran-4-1; SuSy,

sucrose synthase.

Introduction

The arrival of sucrose at terminal sink organs is a dynamic

process that can take a symplastic and/or an apoplastic route

depending on the type of organ and developmental state

(Patrick 1997). In either case, the overall ability of the hetero-

trophic organ to attract photoassimilates is strongly dependent

on the capacity of individual cells to import, metabolize and

store sucrose, ultimately determining plant productivity and

crop yield (Ho 1988).

In organs where apoplastic sucrose unloading occurs, dif-

ferent routes can be envisaged for the subsequent uptake into

storage cells: (i) cleavage of sucrose by an apoplastic invertase

and subsequent uptake of glucose and fructose (Shannon 1972,

Lalonde et al. 1999, Fernie et al. 2000, Oliveira et al. 2002,

Weschke et al. 2003); and/or (ii) import of sucrose by plasma-

lemma-bound carriers (Tegeder et al. 1999, Barker et al. 2000,

Noiraud et al. 2000, Weschke et al. 2000, Manning et al. 2001,

Aoki et al. 2002, Kühn et al. 2003) (Fig. 1 and Supplementary

Fig. 1). In addition, and as evidenced by recent studies using

citrus juice cells and suspension-cultured cells of sycamore

(Acer pseudoplatanus L.), sucrose can also be taken up by

endocytosis and transported to the central vacuole (Etxeberria

et al. 2005a, Etxeberria et al. 2005b). Subsequent conversion of

internalized sucrose to starch involves a series of enzymatic

reactions wherein sucrose synthase (SuSy) (EC 2.4.1.13) pre-

dominantly controls sucrose breakdown and production of a C-

6 starch precursor molecule entering the amyloplast (Keeling et

al. 1988, Appeldoorn et al. 1997, Hatzfeld and Stitt 1990,

Baroja-Fernández et al. 2003). Because of its importance in the

sucrose–starch conversion process, SuSy is considered to be a

major determinant of sink strength in heterotrophic organs

(Geigenberger and Stitt 1993, Martin et al. 1993, Fu and Park

1995, Zrenner et al. 1995, Ruan et al. 2003)

In addition to its role as the major transported assimilate,

there is now compelling evidence that sucrose is an important

mobile signal that regulates a variety of different genes and

processes in various plant organs (Chiou and Bush 1998,

Sokolov et al. 1998, Lalonde et al. 1999, Barker et al. 2000,

Smeekens 2000, Yang et al. 2004). In heterotrophic cells, for

instance, numerous genes and metabolic pathways involved in

starch biosynthesis are induced or stimulated by sucrose (Fu et

al. 1995, Geiger et al. 1998, Müller-Röber et al. 1990, Fernie et

al. 2001), as is the endocytic system for sucrose uptake

(Etxeberria et al. 2005a).

The existence of two entry points for sucrose into storage

cells may represent the channeling of sucrose to different meta-

* Corresponding author: E-mail, javier.pozueta@unavarra.es; Fax: +34-948232191.

1

Endocytic sucrose uptake and starch biosynthesis2

bolic events, with both mechanisms contributing separately to

the overall sink strength. As a first step to investigate to what

extent endocytic sucrose uptake is involved in the control of

both sink strength and starch biosynthesis in heterotrophic

cells, we employed the well characterized sycamore cultured

cells (Rébeillé et al. 1985, Frehner et al. 1990, Pozueta-Romero

et al. 1991, Etxeberria et al. 2005a), potato (Solanum

tuberosum L.) tuber slices (Viola and Davies 1991, Geiger et

al. 1998, Fernie et al. 2001) and developing barley (Hordeum

vulgare) endosperms. In addition, we employed the potent

endocytic inhibitors wortmannin (Emans et al. 2002) and 2-(4-

morpholynyl-)-8-phenyl-4H-1 benzopyran-4-1 (LY294002)

(Vlahos et al. 1994), which do not exert any inhibitory effect

on electrogenic sucrose transport (Etxeberria et al. 2005a), and

latrunculin B (LatB). The results presented in this work provide

a first indication that an important pool of apoplastic sucrose

must be taken up by endocytosis prior to its subsequent conver-

sion into starch in heterotrophic organs of both mono- and

dicotyledonous plants.

Fig. 1 Suggested metabolic pathways for the conversion of apoplastic sucrose to starch in heterotrophic cells involving plasmalemma-bound

carriers as well as vesicle-mediated sucrose transport between the apoplast and the vacuole (Etxeberria et al. 2005a). Apoplastic sucrose can be

converted into glucose and fructose by means of apoplastic invertase. Hexoses enter the cell by plasmalemma-bound carriers and/or diffusion

(Stubbs et al. 2004), and are converted into sucrose (Shannon 1972, Geiger et al. 1998, Fernie et al. 2000, Fernie et al. 2002). Alternatively,

sucrose can be taken up from the apoplast by means of plasmalemma-bound sucrose carriers and/or endocytosis to be transported to the cytosol or

to the vacuole, respectively. Vacuolar sucrose can be exported to the cytosol by means of a tonoplast-bound sucrose transporter (Getz 1991, Keller

1992). Cytosolic sucrose is then converted into ADPglucose and fructose by means of soluble SuSy or membrane-bound SuSy (Amor et al. 1995,

Carlson and Chourey 1996, Baroja-Fernández et al. 2003, Etxeberria and Gonzalez 2004, Muñoz et al. 2005). According to this suggested model,

SuSy catalyzes the production of cytosolic ADPglucose entering the amyloplast by means of an ADPglucose translocator (Pozueta-Romero et al.

1991, Shannon et al. 1996, Shannon et al. 1998, Patron et al. 2004), whereas ADPglucose pyrophosphorylase is involved in the scavenging of

starch breakdown products (Pozueta-Romero et al. 1999, Baroja-Fernández et al. 2004, Baroja-Fernández et al. 2005, Muñoz et al. 2005). Mem-

brane recycling takes place essentially as described by Echeverria (2000). A model involving the production of UDPglucose from sucrose by

SuSy and its stepwise conversion into hexose phosphates and ADPglucose is illustrated in Supplementary Fig. 1. Whether sucrose occurring in

vesicles can be transported to the cytosol by means of hypothetical membrane-bound sucrose carriers is being currently investigated.

Endocytic sucrose uptake and starch biosynthesis 3

Results and Discussion

Starch accumulation is strongly reduced by endocytic inhibitors

in sycamore cells cultured with sucrose

By employing both sycamore cultured cells and citrus

juice cells, as well as the potent endocytic inhibitors wortman-

nin and LY294002, we recently provided strong evidence that

endocytosis is an important mechanism for sucrose uptake in

heterotrophic cells (Etxeberria et al. 2005a). Time course anal-

yses of sucrose accumulation in walled cells revealed a two-

phase process involving an initial 120 min period of carrier-

mediated sucrose uptake, followed by a prolonged phase of

rapid sucrose accumulation that was greatly inhibited by the

endocytic inhibitors wortmannin and LY294002 (cf. Fig. 1,

Etxeberria et al. 2005a).

To investigate whether sucrose taken up by endocytosis is

involved in the starch biosynthetic process, a time course anal-

ysis of starch accumulation was performed using cultured cells

of sycamore previously starved for 24 h prior to re-introduc-

tion of sucrose both in the presence and in the absence of either

wortmannin or LY294002. The rationale behind this experi-

mental approach was that, if endocytic uptake of sucrose is

unimportant in the overall sucrose–starch conversion process,

the rate of starch accumulation in cells cultured in the presence

of endocytic inhibitors would not be affected when compared

with control cells. Conversely, if sucrose linked to starch bio-

synthesis enters via endocytosis, cells cultured in the absence

of endocytic inhibitors will accumulate starch more rapidly

than cells cultured in the presence of endocytic inhibitors.

As illustrated in Fig. 2, the rate of starch accumulation in

cells cultured with 50 mM sucrose was approximately 3 µmol

of glucose transferred to starch g–1 FW during the initial 90 min

of culture, followed by an approximately 30 min asymptotic

phase of low starch accumulation. Similar to the time course

pattern of sucrose incorporation in sycamore cells (cf. Fig. 1,

Etxeberria et al. 2005a), starch accumulation during this initial

120 min culture period was insensitive to endocytic inhibitors,

indicating that sucrose uptake linked to the initial stages of

starch biosynthesis in starved cells does not take place by endo-

cytosis but presumably by plasmalemma-bound carriers. Then

starch accumulation entered a second prolonged phase where

starch accumulated at a rate of 1.5 µmol glucose transferred to

starch g–1 FW h–1. This phase was strongly repressed by endo-

cytic inhibitors, the rate of starch accumulation declining to

250 nmol glucose transferred to starch g–1 FW h–1 in the pres-

ence of either 500 nM wortmannin or LY294002. The overall

data thus suggest that, after a 2 h culture period, an important

pool of sucrose linked to starch biosynthesis enters the cell via

endocytosis in starved cells. Strongly substantiating these

observations was the sharp reduction in the rate of starch accu-

mulation when wortmannin was added after 90 min of culture,

a time when sucrose starts entering the cell by endocytosis (cf.

Fig. 1, Etxeberria et al. 2005a).

Endocytic inhibitors lead to reduced intracellular levels of

ADPglucose, but not of hexose phosphates

We measured the intracellular levels of metabolites

involved in the metabolism of both starch and sucrose in cells

cultured with sucrose both in the presence and in the absence of

500 nM LY294002. As shown in Table 1, analyses of the intra-

cellular levels of sugars after 5 h of culture revealed no signifi-

cant effect of LY294002 on the levels of hexoses and hexose

phosphates. Subsequent time course analysis confirmed that

LY294002 does not exert any significant effect on the intra-

cellular levels of hexoses and hexose phosphates during 6 h of

culture (not shown). In clear contrast, however, intracellular

levels of sucrose, starch, UDPglucose and the starch precursor

molecule ADPglucose in cells cultured with LY294002 were

significantly lower than those of control cells.

We then performed a time course analysis of sucrose accu-

mulated and intracellular ADPglucose content in cells previ-

ously starved for 24 h prior to re-introduction of sucrose both

in the presence and in the absence of LY294002. As illustrated

in Fig. 3, this analysis revealed that, similarly to sucrose, ADP-

glucose accumulation follows a two-phase process involving

an initial 120 min LY294002-insensitive period, followed by a

prolonged phase of ADPglucose accumulation which was

inhibited by LY294002. This result strongly indicates that

ADPglucose biosynthesis is intimately linked to the metabo-

lism of sucrose taken up by endocytosis. Essentially similar

Fig. 2 Time course of the starch accumulated by sycamore cells cul-

tured with 50 mM sucrose in the absence (filled squares) or in the

presence of either 500 nM wortmannin or 500 nM LY294002. Open

squares and open circles represent starch accumulated by cells grown

in the continuous presence of wortmannin and LY294002, respec-

tively. Open triangles represent starch accumulated by cells to which

wortmannin was added after 90 min of culture. In every case, 4-day-

old cultured cells were placed in starving medium for 24 h prior to

addition of sucrose. After sucrose addition, cell aliquots were taken at

the indicated times, washed, extracted in ethanol, and analyzed for

starch content. Values represent the difference between starch content

at the indicated culture time and the starch content at zero time of cul-

ture. Starch content at zero time was 12.5 µmol glucose g–1 FW.

Results are given as the mean ± SE of four analyses per time.

Endocytic sucrose uptake and starch biosynthesis4

results were obtained when investigating the effect of

LY294002 on the intracellular levels of UDPglucose. How-

ever, as illustrated in Supplementary Fig. 2, reduction of

UDPglucose levels by LY294002 was less pronounced than

that of ADPglucose intracellular levels.

The fact that hexose phosphate levels are normal in cells

cultured in the presence of LY294002, whereas sucrose, ADP-

glucose, UDPglucose and starch levels are significantly lower

in cells cultured in the presence of LY294002 than in control

cells indicates that (i) metabolism of sucrose taken up by endo-

cytosis is closely connected to that of both ADPglucose and

UDPglucose, most probably by means of SuSy; (ii) the hexose

phosphate pool is not directly connected to that of sucrose

taken up by endocytosis, but most probably it is connected to

the pool of sucrose and/or hexoses taken up by plasmalemma-

bound carriers; and (iii) hexose phosphates are not intermedi-

ates in the sucrose–starch conversion process (Baroja-

Fernández et al. 2003). In addition, the fact that the effect of

LY294002 on the UDPglucose levels was less pronounced than

on ADPglucose levels suggests that the UDPglucose pool is

not solely connected to sucrose taken up by endocytosis, but

also to hexose phosphates, presumably by means of UDPglu-

cose pyrophosphorylase (EC 2.7.7.9).

Reduction of both ADPglucose levels and rate of starch accu-

mulation by endocytic inhibitors is not accompanied by

changes in activities of enzymes directly connected to starch

and sucrose metabolism

Whether reduction of both ADPglucose and starch levels

by inhibitors of endocytic sucrose uptake was a secondary

effect of arrested enzyme activities was investigated by meas-

uring maximum catalytic activities of a broad range of

enzymes closely connected to starch and sucrose metabolism.

As shown in Table 2, these analyses revealed no significant

changes in ADPglucose pyrophosphorylase (EC 2.7.7.27),

alkaline pyrophosphatase (EC 3.6.1.1), UDPglucose pyro-

phosphorylase, phosphoglucomutase (EC 2.7.5.1), SuSy,

sucrose phosphate synthase (EC 2.3.1.14), acid invertase (EC

3.2.1.26), total starch synthase (EC 2.4.1.21), starch phospho-

rylase (EC 2.4.1.1) and total amylolytic activity between cells

cultured in the presence or absence of wortmannin and

Table 1 Metabolite levels in sycamore cells cultured with

50 mM sucrose in the presence and in the absence of 500 nM

LY294002

Cells were harvested after 5 h of culture. The results are given as the

mean ± SE of extracts from four cultures. Metabolite levels are given

as nmol g–1 FW. Starch content and starch accumulated after 5 h of

culture are given as nmol glucose g–1 FW. ‘Accumulated starch’ is the

difference between starch content after 5 h of culture and the starch

content at zero time. Starch content at zero time (upon addition of

sucrose to the culture medium) was 13,900 ± 1,300 nmol glucose

g–1 FW. ‘Accumulated sucrose’ is the difference between sucrose

content after 5 h of culture and the sucrose content at zero time. Sucrose

content at zero time was 9,000 nmol g–1 FW. Values significantly

different from those of control cells are indicated in bold.

Metabolite (nmol g–1 FW)

Treatment

Control +LY294002

ADPglucose 8.0 ± 0.2 5.0 ± 0.8

UDPglucose 481 ± 20.9 401 ± 7.0

Glucose-6-phosphate 343 ± 20.5 338 ± 19.9

Glucose-1-phosphate 126 ± 20.0 152 ± 21.0

Fructose-6-phosphate 120 ± 18.9 95 ± 23.5

Glucose 3,880 ± 410 3,400 ± 480

Fructose 5,300 ± 1,170 4,980 ± 1,270

Accumulated sucrose 19,600 ± 940 10,800 ± 720

Starch 22,500 ± 1,600 15,700 ± 1,400

Accumulated starch 8,600 ± 530 1,900 ± 470

Fig. 3 Time course of (A) accumulated sucrose and (B) intracellular

ADPglucose content in sycamore cells cultured with 50 mM sucrose in

the absence (filled squares) or in the presence (open circles) of 500 nM

LY294002. ‘Accumulated sucrose’ is the difference between sucrose

content at the indicated culture time and the sucrose content at zero

time of culture. Sucrose content at zero time was 9 µmol g–1 FW. The

time course of UDPglucose is shown in Supplementary Fig. 2. Experi-

mental conditions are the same as those of Fig. 2. Results are given as

the mean ± SE of four analyses per time point.

Endocytic sucrose uptake and starch biosynthesis 5

LY294002. Therefore, the reduction of both ADPglucose and

starch levels in cells cultured with sucrose in the presence of

wortmannin and LY294002 is ascribable to the inhibition of the

endocytic uptake of sucrose and not to a reduction in the activi-

ties of enzymes of sucrose–starch conversion.

Starch accumulation from glucose is unaffected by endocytic

inhibitors

Glucose enters the heterotrophic cell by either carrier-

mediated transport, diffusion or endocytosis (Stubbs et al.

2004, Etxeberria et al. 2005c). Subsequent conversion of the

internalized glucose to starch involves a series of enzymatic

reactions in the cytosol and the amyloplast which are common

to those involved in the sucrose–starch conversion (Geiger et

al. 1998, Sokolov et al. 1998, Fernie et al. 2001). To investi-

gate further whether reduction of both ADPglucose and starch

levels by inhibitors of endocytic sucrose uptake was a second-

ary effect of arrested gluconeogenic machinery, and to investi-

gate whether an important pool of glucose destined to starch

production is also taken up by endocytosis, we compared rates

of starch accumulation in cells cultured with glucose both in

the presence and in the absence of either 500 nM wortmannin

or LY294002.

As illustrated in Fig. 4, sycamore cells cultured with glu-

cose accumulated starch at a rate comparable with cells cul-

tured with sucrose (Fig. 2). In addition, the glucose–starch

conversion process was unaffected by the endocytic inhibitors,

indicating that, in contrast to sucrose, the bulk of glucose des-

tined to starch biosynthesis enters the cell by plasmalemma-

bound carriers and/or diffusion (Stubbs et al. 2004). Further-

more, the data confirm that endocytic inhibitors do not nega-

tively affect the enzymatic machinery involved in starch

biosynthesis.

Rate of starch accumulation is highly reduced by endocytic

inhibitors in potato tuber discs and developing barley endo-

sperms

Having shown that a large proportion of apoplastic

sucrose linked to starch biosynthesis is taken up by endo-

cytosis in sycamore cells, we proceeded to investigate whether

this process also occurs in other species. Towards this end, we

performed a time course analysis of starch production in both

potato tuber discs and developing barley endosperms cultured

with radiolabeled sucrose in the presence and in the absence of

endocytic inhibitors as indicated in Materials and Methods.

Table 2 Enzyme activities in sycamore cells after 5 h of culture with 50 mM sucrose in the pres-

ence and in the absence of 500 nM of endocytic inhibitors

The results are given as the mean ± SE of extracts from four cultures per treatment. Enzyme activities are

given in mU g–1 FW.

Enzyme Control + Wortmannin + LY294002

ADPglucose pyrophosphorylase 27.8 ± 1.4 29.3 ± 1.0 23.1 ± 1.3

UDPglucose pyrophosphorylase 131.8 ± 11.5 124 ± 5.9 117 ± 6.9

Phosphoglucomutase 350 ± 15 382 ± 29 320 ± 28.9

Total amylolitic activity 3,070 ± 363 3,030 ± 346 3,101 ± 422

Starch phosphorylase 26.3 ± 1.4 22.0 ± 3.1 30.9 ± 3.7

Acid invertase 317 ± 22 289 ± 18 301 ± 36

Starch synthase 10.8 ± 0.9 9.1 ± 1.4 10.1 ± 1.1

ADPglucose producing SuSy 392 ± 22 353 ± 41 401 ± 23

Alkaline pyrophosphatase 1,857 ± 170 1,669 ± 209 1,844 ± 61

Sucrose phosphate synthase 1,640 ± 111 1,192 ± 214 1,834 ± 214

Fig. 4 Time course of the starch accumulation in sycamore cells cul-

tured with 50 mM glucose in the absence of endocytic inhibitors (filled

squares) or in the presence of either 500 nM wortmannin (open

squares) or 500 nM LY294002 (open circles). Four-day-old cultured

cells were placed in starving medium for 24 h prior to addition of glu-

cose. After glucose addition, cell aliquots were taken at the indicated

times, washed, extracted in ethanol and analyzed for starch content.

Values represent the difference between starch content at the indicated

culture time and the starch content at zero time. Starch content at zero

time was 12.5 µmol glucose g–1 FW. Results are given as the mean ±

SE of four analyses per time point.

Endocytic sucrose uptake and starch biosynthesis6

As illustrated in Fig. 5, control rates of starch accumula-

tion in developing barley endosperms and potato tuber discs

were 65 and 5 nmol glucose transferred to starch g–1 FW h–1,

respectively. Remarkably, and essentially identically to the case

of cultured cells of sycamore (Fig. 2), rates of starch accumula-

tion were markedly reduced when endocytic inhibitors were

included in the culture medium. The overall data are thus a first

indication that a sizable pool of sucrose linked to starch bio-

synthesis is taken up by endocytosis in different heterotrophic

organs of both mono- and dicotyledonous species.

Our results are consistent with the idea that cells incorpo-

rate sucrose from the apoplasm for its subsequent conversion

into starch. Although there is evidence that sucrose unloading

into parenchyma cells of some sink tissues proceeds symplasti-

cally (Patrick 1997, Viola et al. 2001), an important contribu-

tion of apoplastic sucrose unloading has been underlined by the

facts that (i) expression of a yeast invertase in the apoplasm of

potato tubers is accompanied by important metabolic changes

(Sonnewald et al. 1997, Fernie et al. 2000); and (ii) over-

expression of sucrose transporters in both developing potato

tubers and pea cotyledons results in marked shifts in metabo-

lite levels and/or growth (Rosche et al. 2002, Leggewie et al.

2003).

Additional remarks

Whereas much has been written about the enzymatic path-

ways involved in both sink strength and the sucrose–starch

conversion process in heterotrophic organs, the possible

involvement of different mechanisms of apoplastic sucrose

transport into the cell has not been considered as part of the

overall regulatory system. Our data offer the first direct evi-

dence indicating that, essentially as illustrated in Fig. 1, and as

inferred from the results of Table 1 and Fig. 2 and 5 showing

that endocytic inhibitors prevent the sucrose–starch conversion

process, apoplastic sucrose linked to starch biosynthesis enters

the cell through two entirely different routes: by means of

plasmalemma-bound carriers and by endocytosis. That both

wortmannin and LY294002 strongly inhibit the sucrose–starch

conversion process (Fig. 2) is consistent with previous reports

showing that, although apoplastic glucose can be converted

into starch (Geiger et al. 1998) (Fig. 4), cleavage of sucrose by

apoplastic invertase and subsequent import of glucose into the

cell plays a minor role in the overall starch biosynthetic process

of some heterotrophic organs (Ross et al. 1994, Appeldoorn et

al. 1997, Hajirezaei et al. 2000).

The results presented in this work are consistent with the

view that, at least in some heterotrophic organs, SuSy predomi-

nantly controls sucrose breakdown and production of precur-

sor(s) for starch biosynthesis (Zrenner et al. 1995), whereas

apoplastic invertase catalyzes the hydrolytic breakdown of

sucrose necessary for processes such as phloem unloading, cell

growth and differentiation, osmoregulation, defense response,

etc. (Miller and Chourey 1992, Sonnewald et al. 1997,

Tauberger et al. 1999, for a review see Sturm and Tang 1999).

In addition, and in agreement with the idea that apoplastic su-

crose may act as a signal that regulates carbohydrate metabo-

lism (Lalonde et al. 1999, Smeekens 2000, Fernie et al. 2001),

combined data of Fig. 2 and 4 strongly indicate that conver-

sion of apoplastic glucose into starch is prevented by sucrose.

It is important to point out that, as discussed by Etxeberria

et al. (2005a), endocytic uptake of apoplastic sucrose and its

subsequent conversion to starch does not conflict with transport

through membrane-bound carriers. Our data using starved syc-

amore cells indicate that carrier-mediated sucrose import may

play a crucial role in the overall sucrose–starch conversion

process during the initial period of sucrose unloading (i.e.

during the initial part of the day), whereas endocytic uptake

may play an important role in transporting the bulk of sucrose

to the vacuole after a prolonged period of sucrose unloading.

Fig. 5 Time course of the starch accumulation in developing barley

endosperms and potato tuber discs cultured with 100 mM [U-14C]sucrose (5×105 dpm/µl), in the absence of endocytic inhibitors

(filled squares) or in the presence of either 500 nM wortmannin (open

squares) or 500 nM LY294002 (open triangles). Aliquots were taken at

the indicated times, washed, extracted in ethanol, and incorporated

radioactivities were measured as described in Materials and Methods.

Results are given as the mean ± SE of four analyses per time point.

Endocytic sucrose uptake and starch biosynthesis 7

Tonoplast-bound sucrose carriers (Keller 1992) and SuSy

(Etxeberria and Gonzalez 2004) would subsequently mediate

export of vacuolar sucrose and conversion to starch intermedi-

ates. Whether endocytic vesicles are equipped to export

sucrose to the cytosol is still a matter of conjecture, which is

presently under investigation.

Repression of starch biosynthesis by inhibitors of endo-

cytic sucrose uptake was not limited to compounds interfering

with phosphatidylinositol 3-kinase such as wortmannin and

LY294002 (Li et al. 1995, Brunskill et al. 1998). The F-actin-

depolymerizing drug LatB, which blocks the internalization of

fluid-filled endocytic vesicles (Baluska et al. 2004), markedly

reduced the endocytic uptake of both Lucifer yellow- CH (LY)

(Fig. 6A) and sucrose (Fig. 6B) in starved sycamore cells. Most

importantly, and as illustrated in Fig. 6C, sucrose–starch con-

version was repressed after 2 h of culture with 5 µM LatB. The

overall data thus further strengthen the view that a sizable pool

of sucrose linked to starch biosynthesis is taken up by endo-

cytosis.

The fact that endocytic inhibitors lead to reduced levels

of UDPglucose (Table 1 and Supplementary Fig. 2) appears

to indicate that, in addition to its involvement in the sucrose–

starch conversion process, endocytic sucrose uptake may be

an important determinant of UDPglucose-dependent gluco-

neogenic processes such as synthesis of cell wall poly-

saccharides, glycoproteins, etc. (Amor et al. 1995, Muñoz et al.

1996, Buckeridge et al. 1999, Delmer 1999, Ruan et al. 2003,

Konishi et al. 2004). Further studies will be necessary to

confirm this idea.

Materials and Methods

Plant material

Sycamore (A. pseudoplatanus L) cells were cultured in a medium

supplemented with 50 mM sucrose as described by Frehner et al.

(1990) in continuously agitated 250 ml flasks (200 rpm at 28°C). The

cells were thoroughly washed with ‘starving medium’ (culture

medium without sucrose) and placed back in 250 ml of the same starv-

ing solution. After 24 h starvation, cells were transferred to culture

medium containing either 50 mM sucrose or 50 mM glucose in the

presence or absence of 0.5 µM wortmannin (W-1628, Sigma-Aldrich,

St Louis, MO, USA) or 0.5 µM LY294002 (L-9908, Sigma-Aldrich).

Cell aliquots were taken at the indicated times, washed with starving

medium, filtered through Miracloth and ground to a fine powder in liq-

uid nitrogen. Barley (cv. Scarlett) plants were grown in the fields of

the Public University of Nafarroa. Potato (cv. Desirée) plants were cul-

tivated in growth chambers in individual pots under a 16 h light

(21°C)/8 h dark (16°C) regime.

Enzyme assays

All enzymatic reactions were performed at 37°C. Cultured cells

of sycamore were harvested, washed with starving medium, filtered

through Miracloth and immediately freeze-clamped and ground to a

fine powder in liquid nitrogen with a pestle and mortar. To assay

enzyme activity, 1 g of the frozen powder was resuspended at 4°C in

5 ml of 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithio-

threitol (DTT). The suspension was then desalted and assayed for

Fig. 6 LatB inhibits the endocytic uptake of sucrose linked to starch

biosynthesis. (A) Time course of incorporation of the endocytic

marker LY in sycamore cells cultured in the absence (filled squares) or

in the presence (open circles) of 5 µM LatB. (B) Time course of

sucrose incorporation in sycamore cells cultured in the absence (filled

squares) or in the presence (open circles) of 5 µM LatB. (C) Time

course of the starch accumulated by sycamore cells cultured in the

absence (filled squares) or in the presence (open circles) of 5 µM

LatB. Cells were cultured in starving medium prior to the simultane-

ous addition of both 50 mM sucrose and 1 mM LY. Aliquots were

taken at the indicated times and protoplasts were prepared for LY

measurement. Results are given as the mean ± SE of three analyses per

time point.

Endocytic sucrose uptake and starch biosynthesis8

enzymatic activities. The following enzymes were assayed as

described by Baroja-Fernández et al. (2004): ADPglucose pyro-

phosphorylase, ADPglucose producing SuSy, acid invertase, UDPglu-

cose pyrophosphorylase, alkaline pyrophosphatase, total starch

synthase, starch phosphorylase, total amylolytic activity, phosphoglu-

comutase and sucrose phosphate synthase. One unit of enzyme activ-

ity is defined as the amount of enzyme that catalyzes the production of

1 µmol of product min–1.

Determination of sugars

Cultured cells of sycamore were harvested and immediately

ground to a fine powder in liquid nitrogen with a pestle and mortar. A

0.5 g aliquot of the frozen powder was resuspended in 0.4 ml of 1.4 M

HClO4, left at 4°C for 2 h and centrifuged at 10,000×g for 5 min. The

supernatant was neutralized with K2CO

3 and centrifuged at 10,000×g.

ADPglucose content in the supernatant was determined by using either

one of the following methods: (i) assay A by HPLC on a system

obtained from P.E. Waters and Associates fitted with a Partisil-10-

SAX column as described by Sweetlove et al. (1996); or (ii) assay B

by HPLC with pulsed amperometric detection on a DX-500 system

(Dionex) fitted to a CarboPac PA10 column.

To confirm further that measurements of ADPglucose were cor-

rect, ADPglucose eluted from either the Partisil-10-SAX or CarboPac

PA10 columns was enzymatically hydrolyzed with purified Escherichia

coli ADP-sugar pyrophosphatase (Moreno-Bruna et al. 2001) and

assayed for conversion into AMP and glucose-1-phosphate as

described by Muñoz et al. (2005).

Glucose, sucrose, fructose, glucose-1-phosphate, fructose-6-

phosphate and glucose-6-phosphate were determined by HPLC with

pulsed amperometric detection on a DX-500 system as described by

Muñoz et al. (2005). Starch was extracted and measured by using an

amyloglucosydase-based test kit as described by Baroja-Fernández et

al. (2004).

Labeling experiments

Tuber discs (diameter of 8 mm, thickness of 2 mm) were cut

directly from growing tubers attached to the mother plant and washed

three times in a 20-fold excess of medium containing 10 mM HEPES-

KOH buffer (pH 7.0) and 100 mM sucrose (Viola and Davies 1991).

Both tuber discs and barley endosperms at nearly 15 d after pollina-

tion (eight discs and 30 endosperms in a volume of 5 ml in a 100 ml

Erlenmeyer flask) were incubated in medium including [U-14C]sucrose

(5×105 dpm/µl) (Amersham, UK), shaken at 90 rpm, harvested at the

indicated incubation periods, washed three times in abundant medium

to remove unabsorbed sucrose and debris, frozen in liquid nitrogen and

homogenized in a mortar. The powder thus obtained was washed

thoroughly with abundant 75% methanol/1% KCl, and 14C incorpora-

tion into starch was determined as described by Viola and Davies

(1991). In all cases, >90% of the radiolabel released after digesting

starch with amyloglucosydase was recovered as glucose following

HPLC separation.

Uptake of LY

Four-day-old walled sycamore cells were starved for 24 h. At

this time, the culture medium was replaced with fresh medium con-

taining 50 mM sucrose and 1 mg/ml LY (Molecular Probes, Eugene,

OR, USA) both in the presence and in the absence of 5 µM LatB (Cal-

biochem, Germany). Aliquots were taken at the indicated times and

protoplasts prepared as described by Frehner et al. (1990). From the

protoplast preparation, aliquots were taken and LY was analyzed

fluorometrically in a Fluoroskan Neonat (Labsystems, Hampshire,

UK) with an excitation/emission of 425/550 nm, respectively.

Supplementary material

Supplementary material mentioned in the article is available to

online subscribers at the journal website www.pcp.oupjournals.org.

Acknowledgments

This research was partially supported by the grant BIO2004-

01922 from the Comisión Interministerial de Ciencia y Tecnología and

Fondo Europeo de Desarrollo Regional (Spain). M.T.M.-Z. acknowl-

edges the Spanish Ministry of Culture and Education for a pre-doctoral

fellowship. E.E. expresses his gratitude to the Spanish Ministry of

Culture and Education, to the Consejo Superior de Investigaciones

Científicas and to the Public University of Nafarroa for their support.

We express our gratitude to Beatriz Zugasti for technical assitance and

help.

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(Received December 5, 2005; Accepted January 17, 2006)