Download Utilization and Transport of Mannitol in Olea

Plant Cell Physiol. 48(1): 42–53 (2007)
doi:10.1093/pcp/pcl035, available online at www.pcp.oxfordjournals.org
ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Utilization and Transport of Mannitol in Olea europaea
and Implications for Salt Stress Tolerance
Carlos Conde 1, Paulo Silva 1, Alice Agasse 1, Rémi Lemoine 2, Serge Delrot 2,
Rui Tavares 1 and Hernâni Gerós 1, *
1
Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
UMR CNRS 6161, Transport des Assimilats, Laboratoire de Physiologie, Biochimie et Biologie Mole´culaires Ve´ge´tales,
Bâtiment Botanique, UFR Sciences, 40 Avenue du Recteur Pineau, 86022 Poitiers Ce´dex, France
2
Introduction
Mannitol is one of the primary photosynthetic products
and the major phloem-translocated carbohydrate in Olea
europaea L., an important crop in the Mediterranean basin.
Uptake of mannitol in heterotrophic cell suspensions
of O. europaea was shown to be mediated by a 1 : 1
polyol : Hþ symport system with a Km of 1.3 mM mannitol
and a Vmax of 1.3 nmol min1 mg1 DW. Dulcitol, sorbitol
and xylitol competed for mannitol uptake, whereas glucose
and sucrose did not. Reverse transcription–PCR (RT–PCR)
performed on mRNA extracted from cultured cells exhibiting
high mannitol transport activity allowed the cloning of
a partial O. europaea mannitol carrier OeMaT1. The Vmax
of mannitol uptake and the amount of OeMaT1 transcripts
increased along with polyol depletion from the medium,
suggesting that the mannitol transport system may
be regulated by its own substrate. Addition of 100–500 mM
NaCl to cultured cells enhanced the capacity of the
polyol : Hþ symport system and the amount of OeMaT1
transcripts, whereas it strongly repressed mannitol
dehydrogenase activity. Measurements of cell viability
showed that mannitol-grown cells remained viable 24 h after
a 250 and 500 mM NaCl pulse, whereas extensive loss of
cell viability was observed in sucrose-grown cells. OeMaT1
transcripts increased throughout maturation of olive
fruits, suggesting that an OeMaT is involved in the
accumulation of mannitol during ripening of olive. Thus,
mannitol transport and compartmentation by OeMaT are
important to allocate this source of carbon and energy, as well
as for salt tolerance and olive ripening.
Polyols (or sugar alcohols), the reduced form of aldoses
and ketoses, can be either cyclic (cyclitols) or linear
(alditols), and are present in all living forms. In some
plant species, polyols are direct products of photosynthesis
in mature leaves, together with sucrose. Mannitol is
the most widely distributed sugar alcohol in nature and
has been reported in 4100 species of vascular plants of
several families, including the Rubiaceae (coffee), Oleaceae
(olive, privet) and Apiaceae (celery, carrot, parsley).
Mannitol is synthesized in mature leaves from mannose6-phosphate, through the combined action of a
NADPH-dependent mannose-6-phosphate reductase and a
mannitol-6-phosphate phosphatase. It is transported to sink
tissues where it can be either stored or oxidized to mannose
by an NAD-dependent mannitol dehydrogenase (MTD)
(reviewed by Stoop et al. 1996, Noiraud et al. 2001b).
In contrast to sucrose and monosaccharide transporters, little is known regarding the identity and regulation of
polyol transporters either in sink or in source tissues in
higher plants. The first cDNA encoding a mannitol
transporter of a higher plant was identified and characterized in celery phloem (Noiraud et al. 2001a). This cDNA
(AgMaT1, Apium graveolens mannitol transporter 1)
was used to establish a heterologous expression system in
yeast cells. In the past 5 years, polyol transporters
from Prunus cerasus (Gao et al. 2003), Plantago major
(Ramsperger-Gleixner et al. 2004), Malus domestica (Watari
et al. 2004) and Arabidopsis thaliana (Klepek et al. 2005,
Schneider et al. 2006) have been characterized.
Mannitol production may confer several potential
advantages including more efficient carbon use (Stoop
et al. 1996), resistance against oxidative stress (Smirnoff and
Cumbers 1989, Williamson et al. 1995, Jennings et al. 1998)
and salt tolerance. Thus, the mannitol concentration in
celery grown in hydroponic nutrient solution progressively
Keywords: Mannitol — Olea europaea — Polyol transport
— Salt stress.
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; FDA, fluorescein diacetate; MTD, mannitol dehydrogenase; mltD, mannitol-1-phosphate dehydrogenase; PI,
propidium iodide; TPPþ, tetraphenylphosphonium; RT–PCR,
reverse transcription–PCR.
*Corresponding author: E-mail, [email protected]; Fax, þ 351-253678980.
42
Mannitol uptake in Olea europaea
increases as the total salinity of the growth solution
increases (Stoop and Pharr 1994). Increased mannitol
accumulation in leaves was also observed in plants irrigated
with 0.3 M NaCl, as a consequence of a massive shift in
partitioning of fixed carbon into mannitol instead of
sucrose (Everard et al. 1994). The strong water stress
tolerance of Fraxinus excelsior is in part related to an
accumulation of malate and mannitol (Guicherd et al.
1997), and, in plants subjected to drought stress, the
mannitol content of the leaf xylem sap increases
(Patonnier et al. 1999). Additional evidence for a role for
mannitol in salinity tolerance was obtained when Nicotiana
tabacum, Populus tomentosa and other plants were genetically engineered to synthesize mannitol through introduction of an Escherichia coli mannitol-1-phosphate
dehydrogenase (mltD), which catalyzes the biosynthesis of
mannitol from fructose, resulting in more salt-tolerant
plants (Tarczynsky et al. 1993, Hu et al. 2005).
In Arabidopsis, mltD gene transfer and expression
enhanced seed germination under salinity conditions
(Thomas et al. 1995).
Olea europaea L. is an evergreen moderately salttolerant tree (Therios and Misopolinos 1988, Rugini and
Fedeli 1990) traditionally cultivated in the Mediterranean
basin. Olives and olive oil play an increasingly important
nutritional role and are an essential part of what is now
widely known as the ‘Mediterranean diet’. Sugars are the
main soluble components in olive tissues and play
important roles, providing energy and acting as precursors
for olive oil biosynthesis. Glucose, fructose and galactose
are the main sugars found in olive pulp, but appreciable
quantities of mannitol are also present (Marsilio et al.
2001). Water deficit, freezing, salinity and air pollution are a
few of the stress factors restricting plant growth, so that
olive productivity at the end of the growing season
expresses only a fraction of the plant’s genetic potential
(Vitagliano and Sebastiani 2002). In this context,
the elucidation of the role of mannitol as carbon and
energy source for plant growth and as a protecting solute
43
against salinity and drought is important for the improvement of yield potential of the plant. The present work
characterizes a mannitol transporter expressed in cultured
cells and intact fruits of O. europaea. For the first time in
plants it is shown that mannitol transport is regulated by
means of salt-mediated changes in the transcription of
mannitol carrier(s). Altogether, the results showed
that transmembrane transport of mannitol is a critical
step in terms of osmotic adjustments and productivity
in O. europaea.
Results
Growth in batch cultures with mannitol
Suspension cell cultures mimic heterotrophic plant
tissues, where carbohydrates are imported from photosynthetically active source tissues, thus making them a
suitable model system to study sugar transport into sink
cells. The capacity of O. europaea cell suspensions to use
sucrose, lactose, glucose, galactose, fructose, mannitol and
glycerol as sole carbon and energy sources was studied
previously (Oliveira et al. 2002). Among these substrates,
only lactose and glycerol were unable to promote cell
growth. Growth of O. europaea cell suspensions with 1%
(w/v) mannitol and its consumption are depicted in Fig. 1A.
To ascertain whether mannitol transport constitutes the
rate-limiting step of the growth of O. europaea cells on
mannitol, the growth parameters were compared with the
maximal capacity of mannitol uptake. The following values
for the maximum specific growth rate (mmax) and yield
coefficient (Y ) were estimated: 0.2 d1 and 0.6 g biomass
g1 mannitol, respectively. From the ratio mmax/Y, a value
for the specific mannitol transfer rate (q) of 1.28 nmol min1
mg1 DW was estimated, similar to the corresponding Vmax
of mannitol transport (Table 1), suggesting that mannitol
uptake is an important metabolic step for the control of cell
growth. Growth of O. europaea cells in a medium containing a mixture of 0.5% (w/v) glucose and 0.5% (w/v)
mannitol is depicted in Fig. 1B. The rate of glucose
Fig. 1 Representative growth curves of O. europaea cells in MS medium with 1% (w/v) mannitol (A) and with 0.5% (w/v) glucose and
0.5% (w/v) mannitol (B).
44
Mannitol uptake in Olea europaea
Table 1 Specific rate of mannitol transfer (q), D-[14C]mannitol uptake and MTD activity in suspension-cultured cells of
O. europaea cultivated with 1% (w/v) mannitol
D-[
q
Mid-exponential
growth phase ([mannitol]medium 0.5%)
Late exponential
growth phase ([mannitol]medium 550.1%)
Salt stressedd
1
14
MTD activityc
C]mannitol uptake
1.40 0.07a (0.25 0.05)b
0.22 0.02
1.28
1.29 0.04b
0.47 0.11
–
1.5 0.08b
0.09 0.01
1
Units are in nmol mannitol min mg DW. Values are the means SD of three independent experiments.
a
[mannitol]ext ¼ 20 mM (Fig. 2B).
b
Vmax of the mannitol:Hþ symport system.
c
Vmax of MTD.
d
24 h after 500 mM NaCl addition, as indicated in Fig. 6.
depletion from the medium was higher than that of
mannitol; after glucose exhaustion (day 8), mannitol
sustained cell growth up to day 12.
1.5
A
1.0
1.5
v
1.0
v (nmol min−1mg−1D.W.)
0.5
0.5
0.0
0.0 0.25 0.50 0.75 1.00
v/[S]
0.0
1.5
B
1.0
1.5
v
Mannitol transport
Transport experiments were conducted in O. europaea
cell suspensions harvested at the end of the exponential
growth phase, after 10–15 d in culture, when the mannitol
concentration had fallen to about 0.05% (w/v).
Initial uptake rates of 0.2–20 mM D-[14C]mannitol followed
Michaelis–Menten kinetics (Fig. 2A), suggesting carriermediated transport. By the application of a computerassisted non-linear regression analysis (GraphPad Prism,
version 4.0) to the data, the following kinetic parameters
were obtained: Km, 1.3 0.15 mM mannitol and Vmax,
1.29 0.04 nmol mannitol min1 mg1 DW.
Cells of O. europaea cultivated with 1% mannitol,
collected at the mid-exponential growth phase when the
mannitol concentration in the medium is approximately
0.5% (w/v) (see Fig. 1A), also displayed the capacity to
transport mannitol; however, the Eadie–Hofstee plot of
14
D-[ C]mannitol initial uptake rates was biphasic (Fig. 2B).
The computer-assisted non-linear regression analysis of
the data agreed with the presence of two distinct transport
modes: saturating transport associated with first
order kinetics. The following values were estimated for
the maximal capacity of saturating transport and for the
diffusion-like component: Vmax, 0.25 0.05 nmol mannitol
min1 mg1 DW; kd, 0.06 0.01 ml min1 mg1 DW.
To study the specificity of the transport system, the
initial uptake rates of 0.1–2 mM D-[14C]mannitol were
estimated in the presence of the following unlabeled
putative competitors: dulcitol, sorbitol, xylitol, myoinositol, glucose, fructose, mannose and sucrose. The
acyclic polyols dulcitol, sorbitol and xylitol behaved as
competitive inhibitors (Fig. 3A), suggesting that they share
the same carrier; glucose and the remaining substrates
(not shown) had no effect on the transport of mannitol,
0.5
1.0
0.5
0.0
0.0
0.25 0.50
v/[S]
0.75
0.0
0
5
10
[D-Mannitol] (mM)
15
20
Fig. 2 Mannitol transport by suspension-cultured cells of
O. europaea cultivated with 1% mannitol as in Fig. 1A. Initial
uptake rates of D-[14C]mannitol, at pH 4.5, by cells collected at the
end of the exponential growth phase ([mannitol]medium 550.1%, w/v)
(A) and at mid-exponential growth phase ([mannitol]medium 0.5%,
w/v) (B). Inserts: Eadie–Hofstee plots of the initial D-[14C]mannitol
uptake rates.
thus appearing not to be recognized by the permease.
Although the identified transport system seems to be
specific for polyols, O. europaea cell suspensions cultivated
in similar conditions are able to transport glucose according
Mannitol uptake in Olea europaea
v (nmol min−1 mg−1 D.W.)
0.75
0.9
A
0.50
0.6
0.25
0.3
0.00
0.0
0.25
0.50
45
0.0
0.0
0.75
B
2.5
5.0
7.5
10.0
v / [S]
14
Fig. 3 Eadie–Hofstee plots of the initial uptake rates, at pH 4.5, of D-[ C]mannitol (A) and D-[14C]glucose (B) by suspension-cultured cells
of O. europaea. Transport was measured in the absence of other sugars or polyols (filled boxes) and in the presence of unlabeled 5 mM
sorbitol (filled inverted triangles), 5 mM dulcitol (filled triangles), 5 mM xylitol (filled diamonds), 20 mM mannitol (open triangles) and
20 mM glucose (open squares). Cells were cultivated with 1% (w/v) mannitol as in Fig. 1A and collected at the end of the exponential
growth phase as described in Fig. 2A.
to a carrier-mediated mechanism with Km ¼ 67 30 mM
glucose and Vmax ¼ 1.45 0.26 nmol glucose min1 mg1
DW (Oliveira et al. 2002). Here, inhibition experiments of
14
D-[ C]glucose uptake by different polyols were performed
to check that polyol uptake does not occur via the
monosaccharide transport system. Mannitol at 20 mM did
not inhibit 0.02–0.5 mM D-[14C]glucose uptake (Fig. 3B).
Similar results were obtained with sorbitol and dulcitol
(data not shown). Taken together, the results suggest
that, in O. europaea, monosaccharides and polyols are
transported via two distinct transport systems with
different Kms. This could account for the results depicted
in Fig. 1B, where glucose is the first substrate to be
consumed when growth occurred in a medium with glucose
and mannitol.
To study the energetics of mannitol transport in
O. europaea cultured cells, D-[14C]mannitol uptake was
measured at different external pH values. Vmax decreased
abruptly from pH 4.5 to 5.5, with little activity
remaining above pH 5.5, consistent with the involvement
of a proton-dependent transport system (Fig. 4A).
Additionally, the uptake of 0.2–2.0 mM [14C]mannitol was
strongly inhibited by 50 mM of the protonophore
m-chlorophenylhydrazone (CCCP) (Fig. 4B). The occurrence of transient alkalinization of extracellular media upon
addition of mannitol to cell suspensions (Fig. 4C) provided
clear evidence for the involvement of a mannitol–proton
symport system. The initial velocities of proton uptake were
estimated from the slope of the initial part of the pH trace
(the alkalinization curve) after 5 mM mannitol (saturating
concentrations) had been added. A value of 1.1 nmol Hþ
min1 mg1 DW can be obtained from the data of Fig. 4C,
similar to the maximal capacity of the mannitol : Hþ
symport system measured with D-[14C]mannitol, suggesting
a 1 mannitol : 1 proton stoichiometry. Since such a
mechanism would be associated with a net influx of positive
charges into the cells, the effect of the dissipation of
transmembrane electric potential on mannitol transport was
studied. Fig. 4B shows the effect of the lipophilic cation
tetraphenylphosphonium (TPPþ) on the initial uptake rates
of 0.2–2 mM D-[14C]mannitol. TPPþ inhibited mannitol
uptake, indicating that membrane potential makes a
significant contribution to the driving force for substrate
transport by the polyol carrier.
Polyol : Hþ symport activity and OeMaT expression
As the Vmax of the O. europaea polyol : Hþ symport
system increased along with mannitol depletion from the
medium throughout the exponential growth (see Fig. 2),
mannitol levels appear to have a regulatory effect. To study
the induction of transport activity in response to mannitol
concentration, the accurate dependence of permease activity
on polyol levels in the medium was evaluated. Cells were
grown in a medium with 1% (w/v) mannitol, and the uptake
of D-[14C]mannitol was measured in cell aliquots harvested
from the culture at the times indicated in Fig. 5. When the
external levels of mannitol fell below 0.05% (w/v) (day 13),
the activity of the polyol transport system increased
abruptly from basal levels. Maximal activity was observed
when mannitol was completely exhausted from the culture
medium (day 15). The addition of 0.05 mg ml1 cycloheximide to the culture medium prevented the increase of
mannitol transport activity, suggesting the involvement
of de novo protein synthesis.
To identify the cDNA sequence encoding the
O. europaea mannitol : Hþ symport system, degenerated
primers corresponding to conserved regions of polyol
transporters were used, and reverse transcription–PCR
46
Mannitol uptake in Olea europaea
Vmax
(nmol min−1 mg−1 D.W.)
0.75
A
1.2
1.0
B
0.50
0.8
0.25
0.6
0.4
4.5
5.0
5.5
6.0
6.5
0.00
0.00
7.0
0.25
pH
0.50
0.75
v/(S)
C
Alkalinization
(10 nmol H+)
10 s
2 mM
mannitol
Fig. 4 Energetics of the O. europaea polyol transport system. (A) pH dependence of mannitol transport. (B) Eadie–Hofstee plots of the
initial uptake rates of D-[14C]mannitol in the absence (filled squares) or presence of 50 mM CCCP (open circles) and 10 mM TPPþ
(filled triangles). (C) Proton movements, at pH 4.5, associated with the addition of 2 mM mannitol to cell suspensions. Cells were cultivated
with 1% (w/v) mannitol as in Fig. 1A and collected at the end of the exponential growth phase as described in Fig. 2A.
(RT–PCR) was performed on mRNA extracted from
O. europaea suspension-cultured cells exhibiting high
mannitol transport activity. This allowed the cloning
of a 501 bp cDNA OeMaT1 (accession No. DQ059507)
with extensive homology to the celery mannitol
transporter AgMaT2. Low-stringency Southern blots of
O. europaea genomic DNA digested with four different
restriction enzymes and hybridized with OeMaT1 cDNA,
identified one (HindIII and XhoI) and three (XbaI and
EcoRI) bands (Fig. 5D). This suggests the presence of a
multigene family of polyol transporters in olive tree, which
is consistent with evidence available for other plant species
such as P. cerasus (Gao et al. 2003), P. major (RamspergerGleixner et al. 2004), M. domestica (Watari et al. 2004) and
A. thaliana (Klepek et al. 2005). An OeMaT1 partial cDNA
sequence was used as a probe for Northern analysis
(Fig. 5B). Due to the impossibility of obtaining the
full-length cDNA, we were not able to design a specific
probe. We must therefore consider the possibility of a high
level of sequence identity with the other potential mannitol
transporters present in the O. europaea genome with
the probable occurrence of cross-hybridization. For the
rest of the work, we will refer to OeMaT to indicate that
the signals observed in Northern experiments were either
due to OeMaT1 or to other unidentified mannitol
transporters present in O. europaea. The parallel between
OeMaT transcripts and mannitol transport activity shown
in Fig. 5B suggests that carrier expression is mainly
controlled at the transcriptional level, although other
forms of post-transcriptional regulation cannot be ruled
out. These results show for the first time a tight regulation
of a mannitol : Hþ symport expression by external levels
of its own substrate.
As referred to in the Introduction, substantial amounts
of mannitol are present in the pulp of olive fruit, reaching
maximum values in ripened olives of 8 mg g1 DW
(Marsilio et al. 2001). To study the involvement of the
polyol : Hþ symport system in mannitol unloading during
olive fruit maturation, RNAs were isolated from olive fruits
at the green, cherry and black stages, and the expression of
OeMaT was studied. Although detectable throughout fruit
development, OeMaT transcript levels strongly increased
during the late stage of the ripening process (black stage) at
the onset of mannitol accumulation (Fig. 5C).
Effect of salt stress on polyol : Hþ symport activity and
OeMaT expression
As referred to in the Introduction, mannitol can act as
a compatible solute besides its role as a carbon and energy
source. To study the influence of salt stress on mannitol
( ) Mannitol (%, w/v)
1.2
0.05 mg mL−1 cyclohexamide
Control
0.5
0.4
0.9
0.3
0.6
0.2
0.3
0.1
0.0
Vmax
(nmol min−1 mg−1 D.W.)
A
Vmax
(nmol min−1 mg−1 D.W.)
Mannitol uptake in Olea europaea
A
1.5
10.0
13.0
14.0
14.5
Days after subculture
0 mM NaCl
500 mM NaCl
1.0
0.5
0.0
0.0
7.0
47
0
15.0
6
12
24
Time (h)
B
OeMaT 1
OeMaT1
0 mM NaCl
OeMaT1
500 mM NaCl
rRNA
Vmax
(nmol min−1 mg−1 D.W.)
Bl
ac
k
rry
he
G
re
C
C
en
rRNA
B
OeMaT 1
III
Ec
oR
I
Xh
oI
(bp)
H
in
d
D
Xb
al
rRNA
1.5
1.0
0.5
0.0
0.0
11490
100.0 250.0 300.0
[NaCl] (mM)
500.0
OeMaT1
2560
rRNA
1159
Fig. 5 Mannitol : Hþ symport activity and OeMaT expression in
O. europaea. (A) Cell suspensions were cultivated with 1% (w/v)
mannitol, and D-[14C]mannitol uptake was measured in cell
aliquots harvested from the culture at the times indicated.
Cycloheximide (0.05 mg ml1) was added to a 25 ml aliquot of
the culture at day 14. (B) Northern blot analysis of OeMaT
expression in the suspension-cultured cells in the absence of
cycloheximide and (C) in developing olive fruit at different stages of
olive maturation. Total soluble mannitol concentration in the fruit
pulp (mg g1 DW): green, 5.5; cherry, 5.5; and black, 8.0 (Marsilio
et al. 2001). (D) Southern blot analysis of OeMaT1 in O. europaea
genomic DNA.
transport capacity, NaCl was added to suspension-cultured
cells at mid-exponential growth phase. Addition of 500 mM
NaCl promoted the increase of OeMaT transcription and
mannitol transport activity with time when compared
Fig. 6 Effect of salt stress on O. europaea mannitol : Hþ symport
activity and OeMaT expression. (A) Time dependence of Vmax of
mannitol transport and OeMaT transcripts upon addition of
500 mM NaCl. Cells were grown with 1% (w/v) mannitol as in
Fig. 5, NaCl was added to 25 ml aliquots of the culture at day 8 and
14
D-[ C]mannitol uptake was measured in cell aliquots at the time
periods indicated. (B) Dependence of Vmax of mannitol transport
and OeMaT transcripts on NaCl concentration in the culture
medium. Different NaCl concentrations were added to 25 ml
aliquots of the culture at day 8 as in A, and D-[14C]mannitol uptake
was measured in cell aliquots 24 h after salt addition.
with control cells, the maximal levels being achieved
within 24 h (Fig. 6A). Additionally, the increase of salt
concentration in the medium promoted a dose-dependent
increase of OeMaT transcripts and polyol : Hþ symport
activity, measured after 24 h (Fig. 6B). The parallel between
Vmax of mannitol transport and OeMaT message levels
suggests that the expression of OeMaT is responsible for the
48
Mannitol uptake in Olea europaea
A - Mannitol-grown cells
0 mM NaCl
500 mM NaCl
250 mM NaCl
500 mM NaCl
Visible
0.4
250 mM NaCl
0.005
0.010
0.015
v/[S]
Fig. 7 MTD activity of O. europaea suspension-cultured cells.
Cells were grown with 1% (w/v) mannitol as in Fig. 1 and collected
at mid-exponential growth phase, as described in Fig. 2B
([mannitol]medium 0.5%, w/v) (filled circles) and at the end of
the exponential growth phase, as indicated in Fig. 2A
([mannitol]medium 550.1%, w/v) (filled squares). MTD activity of
salt-stressed cells measured 24 h after 500 mM NaCl addition, as
described in Fig. 6 (filled triangles).
increase of mannitol transport capacity under salt stress
conditions.
Regulation of mannitol catabolism by mannitol
dehydrogenase
The activity of MTD has been fully characterized
during the last decade by Pharr and co-workers in celery,
where mannitol also represents an important carbon and
energy source. Here, MTD activity was measured in crude
extracts of O. europaea cells to correlate mannitol transport
activity with the rate of intracellular mannitol conversion.
Crude extracts were obtained from cells cultivated with
1% (w/v) mannitol up to mid- and late exponential growth
phase as in D-[14C]mannitol transport experiments.
The activity of MTD was also measured in homogenates
from mannitol-grown cells subjected to salt stress.
The Eadie–Hofstee plots of initial velocities of mannitol
oxidation were linear over 5–150 mM mannitol, and a Km of
40 mM was obtained (Fig. 7). MTD activity was enhanced
as mannitol was depleted from the culture medium and
strongly repressed by salt. The decrease of MTD activity in
salt-stressed cells associated with the increase of mannitol
uptake capacity should allow the intracellular accumulation
of mannitol to compensate for the decrease of external
water activity.
Evaluation of the protective role of mannitol against salt
stress
To assess the physiological role of mannitol
in salt stress tolerance in O. europaea, we studied cell
viability after salt addition to mannitol-grown cells and
B - Sucrose-grown cells
0 mM NaCl
Visible
0.0
0.000
Epifluorescence
0.2
Epifluorescence
v (µmol h−1 mg−1 protein)
0.6
Fig. 8 Cell viability assays in O. europaea suspension-cultured
cells cultivated with mannitol (A) and sucrose (B) 24 h after
the addition of 250 and 500 mM NaCl. Fluorescence was measured
after incubation with fluorescein diacetate (FDA, green
fluorescence) and propidium iodide (PI, red fluorescence).
sucrose-grown cells. Cell aliquots were collected from each
medium at mid-exponential growth phase, and 250 and
500 mM NaCl were added. Cell viability was assessed by
fluorescein diacetate (FDA) and propidium iodide (PI). The
intact plasma membrane is permeable to FDA, and FDA is
converted to a green fluorescent dye, fluorescein, by a
function of internal esterases, showing a green color in
viable cells. In contrast, the intact plasma membrane is
impermeable to PI. Damaged cells having pores on the
plasma membrane incorporate the dye, which binds to
genomic DNA and generates a red fluorescence (Jones and
Senft 1985). Fig. 8 shows that a large population of
Mannitol uptake in Olea europaea
mannitol-grown cells remained viable 24 h after a 500 mM
NaCl pulse. Sucrose-grown cells were not able to display
green fluorescence after the same treatment, and showed
extensive loss of cell viability as evaluated by the PI red
fluorescence. A similar result was obtained with glucosegrown cells (not shown). Taken together, these results
suggest that mannitol plays essential roles in O. europaea,
both providing a carbon and energy source for sink
tissues and acting as an osmoprotectant in response to
high salinity.
Discussion
Mannitol is the most abundant carbohydrate in olive
tree leaves, accounting for 82–92% of the total soluble
carbon (Drossopoulos and Niavis 1988), and is an
important sugar in the olive fruit pulp (Marsilio et al.
2001). However, very little information is available regarding the utilization and transport of mannitol in this
polyol-synthesizing plant. In the present work, mannitol
transport mechanisms operating in O. europaea heterotrophic cells were investigated in detail. In addition, the
regulation of the expression of the polyol transport system
and its relevance in osmotic adjustments was also studied.
The data obtained are particularly relevant because the
olive tree is normally cultivated in areas in which water is
the main limiting factor in agricultural production (Tattini
et al. 1994). Although it is recognized that suspensioncultured cells may not be close to normal physiological
conditions, they provide a convenient experimental system
that has already yielded a lot of useful information on sugar
transport mechanisms and regulation (Roitsch and Tanner
1994, Ehness et al. 1997, Oliveira et al. 2002, Cakir et al.
2003, Azevedo et al. 2006, Conde et al. 2006).
Mannitol-grown cells of O. europaea exhibit a specific
growth rate (mmax, 0.2 d1) higher than cells cultivated with
glucose (mmax, 0.07 and 0.11 d1 in media with 0.5 and 3%
glucose, respectively; Oliveira et al. 2002) or 2% sucrose
(mmax, 0.08 d1), possibly because mannitol catabolism
produces a higher number of ATPs than the catabolism of
an equal amount of glucose or sucrose. From the linear part
of the growth curve, a value for the yield coefficient (Y ) of
0.6 g biomass g1 mannitol was estimated, suggesting that
most of the carbohydrate is respired and used as a carbon
and energy source for exponential growth. Therefore, little
or no sugar is channeled to internal stores, which is
confirmed by the arrest of cell growth that was associated
with the decline of sugar content. The fact that mannitol
behaved as an efficient carbon and energy source in
heterotrophic cells of O. europaea is consistent with
the role of this compound as a major photoassimilate
in this species.
49
The saturable transport observed in O. europaea cells
involves a polyol : Hþ symport system with a stoichiometry
of 1 mannitol : 1 Hþ as indicated by the following
observations: (i) mannitol addition to weakly buffered cell
suspensions is associated with a transient alkalinization of
the extracellular medium; (ii) the Vmax of proton uptake is
similar to the Vmax of carrier-mediated D-mannitol uptake
and depended on extracellular pH; (iii) dissipation of the
proton-motive force by CCCP significantly inhibited the
initial velocities of D-mannitol uptake; and (iv) mannitol
transport was sensitive to TPPþ, suggesting that the is
an important component of the proton-motive force
involved in mannitol accumulation. Proton dependence
and substrate affinity (Km, 1.3 mM mannitol) are in good
agreement with the data obtained for the celery mannitol
transporter. In this polyol-producing plant, different
membrane transport steps have been studied, from
phloem loading to phloem unloading and storage in
parenchyma cells. The cloned AgMaT1 gave yeast cells
the ability to grow on mannitol, and a Km value for
mannitol uptake of 0.34 mM was obtained (Noiraud et al.
2001a), that correlates well with the value (Km, 0.64 mM)
determined in plasma membrane vesicles from phloem
strands of celery (Salmon et al. 1995). The involvement of a
co-transport with protons was proposed because the uptake
of mannitol was almost abolished by CCCP and was
maximal at acidic pH. Also, in storage parenchyma discs of
celery leaves and in plasma membrane vesicles from
parenchyma cells, Km values of 1 mM were obtained
(Keller 1991, Salmon et al. 1995). In contrast, mannitol
transport in vacuoles of celery parenchyma cells seems to be
mediated by facilitated diffusion, because it was neither
stimulated by energization with ATP and pyrophosphate,
nor impaired by the dissipation of the proton-motive force
(Greutert et al. 1998).
The selectivity of the O. europaea polyol : Hþ symport
system is rather poor for mannitol because the transport
system was able to accept, besides mannitol, dulcitol,
sorbitol and xylitol, as these acyclic polyols behaved as
competitive inhibitors. In addition, monosaccharide uptake
and polyol uptake are mediated by two distinct transport
systems since glucose did not inhibit D-[14C]mannitol
uptake. Likewise, mannitol did not affect D-[14C]glucose
uptake mediated by a monosaccharide : Hþ symport system
previously identified in mannitol-grown cells (Oliveira et al.
2002). Accordingly, glucose is not transported by
Saccharomyces cerevisiae expressing the celery mannitol
transporter AgMaT1 (Noiraud et al. 2001a). This is in
contrast to the polyol transporter from Arabidopsis
AtPST5 which can also transport glucose (Klepek et al.
2005). Kinetic parameters may explain why D-glucose is
consumed before mannitol when the two substrates are
present in the extracellular medium: the capacity of both
50
Mannitol uptake in Olea europaea
activity increased, suggesting that mannitol regulates
carrier expression at the transcriptional level, although
other levels of regulation cannot be ruled out.
Salt stress can be regarded as a situation in which
plants have to cope with both decreased water availability
and ion toxicity (Lewitt 1980). Despite numerous studies
indicating that, following polyol synthesis in mature leaves,
there is an increase in polyol content in sink organs in
response to drought or salt stress (see Introduction), and
that salt alters enzyme activities related to polyol metabolism (Stoop and Pharr 1994, Williamson et al. 1995), little is
known about the regulation of the expression of polyol
transporters under such conditions. In Mesembryanthemum
crystallinum, where the genes MITR1 and MITR2 behave as
myo-inositol : Naþ symporters, myo-inositol could be a
signal during the adaptation to salt stress (Nelson et al.
1999, Chauhan et al. 2000). In celery plants subjected to
severe salt stress, the expression of AgMaT1 has not been
studied yet, but it appears that the expression of the sucrose
carrier AgSUT1 decreased in all organs (Noiraud et al.
2000), suggesting that transport of mannitol is favored.
The present work provides clear evidence that O. europaea
suspension-cultured cells exposed to high salinity display an
increase of OeMaT expression and mannitol transport
activity, together with a drastic decrease of mannitol
oxidation by MTD. This should allow the intracellular
accumulation of mannitol in order to compensate the
decrease of external water activity, providing a tolerance
mechanism to salinity in O. europaea (Fig. 9). Similarly,
celery plants exposed to high salinity showed a specific
down-regulation of MTD activity in sink tissues, resulting
in decreased mannitol use and an accompanying increase in
mannitol accumulation (Stoop and Pharr 1994, Pharr et al.
1995), and in cell suspensions, Mtd transcripts decreased
in parallel with MTD activity upon addition of NaCl
(Williamson et al. 1995). The present work demonstrates
the importance of mannitol as a carbon and energy source
in O. europaea, as well as its role in osmotic protection.
transport systems is similar but the substrate affinity of the
monosaccharide transport system is much higher than that
of the polyol transport system, Km 0.67 mM and 1.3 mM,
respectively. However, one cannot exclude that the activity
of MTD may be repressed by sugar as was reported
in celery (Prata et al. 1997), impairing the intracellular
conversion of mannitol before glucose depletion.
Since mannitol transport plays a key role in
source–sink interaction in O. europaea, it is likely that the
expression and activity of mannitol transporters are highly
regulated by mannitol levels. Our data indeed show that
in O. europaea suspension-cultured cells, alterations in
mannitol levels have a pronounced effect on the expression
and mannitol transport activity of OeMaT. When high
mannitol is present, energy-independent diffusional uptake
is the preferred mode of mannitol absorption, and OeMaT
expression and transport activity are maintained at basal
levels. Whether non-saturable mechanisms involved in the
diffusional uptake may play important roles in sink cells of
olive in vivo needs further investigation, but it may be
possible owing to the high sugar content in sink tissues
(Patrick 1997). While non-saturable mechanisms of sugar
and polyol transport were also reported in other plant cells
and tissues (Delrot 1989, Keller 1991, Krook et al. 2000,
Oliveira et al. 2002, Conde et al. 2006), its underlying
mechanisms are still poorly understood. Several mechanisms or a combination of them could account for
‘diffusion-like’ kinetics: non-specific permeation of the
sugar by free diffusion across the plasma membrane,
involvement of carrier(s) or channels with very low affinity,
or endocytic processes as reported by Etxeberria et al.
(2005). When external mannitol decreases to residual levels,
the linear transport component no longer sustains mannitol
uptake at a rate sufficient to allow efficient activity
of MTD, which exhibits a quite high Km of 40 mM,
and the involvement of a concentrative, energy-dependent
transport system becomes critical. Following mannitol
depletion, OeMaT transcript levels and polyol : Hþ symport
H+ mannitol
H+ mannitol
H+ mannitol
H+ mannitol
High salinity
H+ mannitol
NAD+
NADH
H+ mannitol
MTD
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
Mannitol
Mannitol
NaCl
Fig. 9
Mannitol
NaCl
H+
MTD
Mannitol
Mannose
Low salinity
H+ mannitol
NaCl
Regulation of mannitol transport and metabolism as a mechanism providing salt tolerance in O. europaea.
NaCl
NaCl
NaCl
Mannitol uptake in Olea europaea
Indeed, after a salt pulse, a high percentage of mannitolgrown cells remained viable 24 h after addition of 250
and 500 mM NaCl, contrasting with the dramatic decrease
of cell viability in sucrose-grown cells. Similarly, the growth
rate of heterotrophic celery cell suspensions cultivated
with sucrose was much more inhibited by NaCl than that
of mannitol-grown cells, although it was demonstrated
that both types of cells accumulated soluble sugars to the
same osmotic potential (Pharr et al. 1995). Growing
plants on soil with high salinity represents a challenge for
the future, and it is therefore important to understand
the strategies used by plants to cope with such stress. Given
the apparent potential of mannitol for osmoprotection,
the engineering of plants with mannitol metabolism is
worth investigating.
Although it is clear that mannitol and sorbitol are
translocated in phloem from their site of synthesis to their
site of use, in a way very similar to sucrose in most plants,
there is some controversy about the pathways involved in
phloem loading and unloading. In the case of mannitol,
unloading pathways have been poorly studied, although
carrier-mediated mannitol uptake had been demonstrated in
tissue discs and vacuoles of storage parenchyma of celery
petioles (Keller and Matile, 1989, Keller 1991, Greutert
et al. 1998). In the present work, we showed that OeMaT is
expressed differentially during olive fruit maturation,
suggesting apoplastic unloading. This is in contrast to the
initial claim that polyol-transporting species such as the
olive tree were symplastic loaders (Flora and Mandore
1993). However, the co-existence of symplastic connections
between the sieve tubes and olive pulp cells cannot be ruled
out. Thus, mannitol transport by OeMaT1 exerts an
important role in the context of olive fruit maturation.
Materials and Methods
Cell suspensions and growth conditions
Cell suspensions of O. europaea L. var. Galega Vulgar were
maintained in 250 ml flasks on a rotatory shaker at 100 r.p.m., in
the dark, at 258C on modified Murashige and Skoog (MS) medium
(Murashige and Skoog 1962), supplemented with 1% (w/v)
mannitol, 0.5% (w/v) mannitol plus 0.5% (w/v) glucose, or 1%
sucrose. Cells were subcultured weekly by transferring 10 ml
aliquots into 70 ml of fresh medium. Growth was monitored as
described previously (Conde et al. 2006).
Transport studies of radiolabeled substrates and proton uptake
Harvested cells were centrifuged, washed twice with ice-cold
culture medium without sugar at pH 4.5, and resuspended in the
same medium at a final concentration of 5 mg DW ml1.
To estimate the initial uptake rates of D-[14C]mannitol, 1 ml of
cell suspension was added to 10 ml flasks, with shaking
(100 r.p.m.). After 2 min of incubation, at 258C, the reaction was
started by the addition of 40 ml of an aqueous solution of
radiolabeled sugar-alcohol at the desired specific activity and
concentration. The specific activities were defined according to the
51
final concentration of the polyol in the reaction mixture,
as follows: 500 d.p.m. nmol1 (0.1–2 mM), 100 d.p.m. nmol1
(5–20 mM). Sampling times were 0, 60 and 180 s, time periods
during which the uptake was linear. Washing, radioactivity
measurements and calculations were performed as described by
Conde et al. (2006).
Competition between labeled substrates and other sugars and
polyols was tested by running competitive uptake kinetics.
Inhibition of D-[1-14C]mannitol transport by non-labeled sugars
and polyols was assayed by adding simultaneously the labeled and
non-labeled substrate. The concentration range of labeled mannitol
varied from 0.1 to 2 mM and the final concentration of the
unlabeled substrate was at least 10-fold higher than the Km value
estimated for the transport system. Competitive inhibition of
14
D-[U- C]glucose transport (0.02–0.5 mM) was studied with 20 mM
of either mannitol, sorbitol or dulcitol.
Mannitol-induced proton uptake in the cells was measured as
described earlier (Conde et al. 2006).
Cloning of a mannitol transporter gene
To identify potential cDNA sequences encoding mannitol
transporters in O. europaea, degenerated primers were designed
based on conserved regions of plant polyol transporters. The
primers were OeY50 [forward, 50 -TTTTAGCTTCAATGA(A/
C)TTC(A/C)-30 ] and OeY30 [reverse, 50 -CAA(C/T)TCTTTCCA
CAC(A/T)GC-30 ]. RT–PCR was performed on RNA extracted
from suspension-cultured cells exhibiting high mannitol transport
activity. The amplified 501 bp cDNA was cloned into the pGEM-T
easy vector (Promega, Madison, WI, USA) according to
the manufacturer’s instructions, sequenced and subsequently
named OeMaT1.
RNA gel blot analysis
Total RNAs from olive fruits, harvested at green, cherry and
black stages of the ripening process, and from olive cell suspension
samples were isolated by phenol extraction and 2 M LiCl
precipitation (adapted from Howell and Hull 1978). RNA blot
analysis was conducted as described in Conde et al. (2006), using a
partial [32P]OeMaT1 probe.
Southern blot analysis
Genomic DNA of O. europaea was isolated from olive fruit
according to Steenkamp et al. (1994). The DNA (10 mg) was
digested with EcoRI, HindIII, XbaI and XhoI restriction enzymes.
Digested genomic DNA was separated by electrophoresis in
a 0.8% agarose gel, and blotted on a Hybond N membrane
(Amersham, Little Chalfont, UK). The membrane was prehybridized for 3 h at 558C in 250 mM sodium phosphate buffer
pH 7.2, 1% bovine serum albumin (BSA), 7% SDS, 1 mM EDTA.
The membrane was hybridized overnight at 558C in the
same buffer containing randomly primed [32P]OeMaT1 probe
(prime-a-gene, Promega). The membrane was washed twice in
2 SSC containing 0.1% SDS for 15 min and once for 5 min
in 0.1 SSC and 0.1% SDS at 558C. The membrane was exposed
to an autoradiographic film and imaged using a Bio-Imaging
analyzer (Bio-Rad personal molecular imager FX).
Determination of cell viability
FDA and PI double staining was used to estimate cell
viability, as described earlier (Jones and Senft 1985).
A concentrated stock solution of FDA (500 mg ml1, Sigma,
St Louis, MO, USA) was prepared in dimethylsulfoxide and of PI
52
Mannitol uptake in Olea europaea
(500 mg ml1, Sigma) in water. For the double staining protocol,
1 ml of cell suspensions was incubated with 10 ml of FDA stock
solution and 1 ml of PI stock solution in the dark for 10 min at
room temperature. Cells were observed under a Leitz Laborlux S
epifluorescence microscope with a 50 W mercury lamp and
appropriate filter settings. Images were acquired with a 3CCD
color video camera (Sony, DXC-9100P), a frame grabber
(IMAGRAPH, IMASCAN/Chroma P) and software for image
management and archiving (AxioVision Version 3.0, Carl Zeiss
Vision, GmbH).
Determination of mannitol dehydrogenase activity
Protein extraction and MTD activity assays were determined
as described by Stoop and Pharr (1993). Olea europaea suspensioncultured cells were harvested as described above and ground in a
chilled mortar using a 1 : 4 (v/v) powder : buffer ratio.
The extraction buffer contained 50 mM MOPS (pH 7.5), 5 mM
MgCl2, 1 mM EDTA, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% (v/v) Triton X-100.
Homogenates were centrifuged at 20,000g for 20 min and the
supernatants were stored on ice. MTD activity was assayed by
monitoring the reduction of NADþ spectrophotometrically at
340 nm. Assays were conducted at room temperature (258C) in a
total volume of 1 ml. The reaction mixture contained 100 mM
Bis-Tris propane (pH 9.0), 2 mM NADþ, enzyme extract, and
D-mannitol at the desired final concentration. The reactions were
initiated by the addition of mannitol. Protein concentrations were
determined by the method of Bradford (1976) using BSA as
a standard.
Acknowledgments
This work was supported in part by the Pessoa Program
(GRICES/EGIDE), the Conférence des Présidents d’Université
(CPU), the Conselho de Reitores das Universidades Portuguesas
(CRUP), the Fundação para a Ciência e a Tecnologia (research
project no. POCI/AGR/56378/2004; grant no. SFRH/BD/10689/
2002 to C.C.; grant no. SFRH/BD/13460/2003 to P.S.; and grant
no. SFRH/PBD/17166/2004 to A.A.).
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(Received September 12, 2006; Accepted November 12, 2006)