Download Protonation and Light Synergistically Convert Plasmalemma Sugar

Plant Physiol. (1981) 67, 1146-1 50
0032-0889/81/67/1 146/05/$00.50/0
Protonation and Light Synergistically Convert Plasmalemma
Sugar Carrier System in Mesophyll Protoplasts to its Fully
Activated Form'
Received for publication September 3, 1980 and in revised form December 10, 1980
MICHA Guy, LEONORA REINHOLD, MICHAELA RAHAT, AND AZA SEIDEN
Department of Botany, The Hebrew University of Jerusalem, Israel
ABSTRACT
The course of sugar fluxes into and out of protoplasts isolated from the
mesophyll of Pisum sativum L. has been followed over brief time intervals
(minutes). Light strongly stimulated net sugar influx at pH 8 as well as at
pH 5.5. The proton conductor carbonyl cyanide m-chlorophenylhydrazone
(CCCP) inhibited initial influx in the light, both at pH 8.0 and at pH 5.5.
CCCP was without effect in the dark at either pH. All these results applied
both to sucrose and to the nonmetabolizable glucose analog 3-0-methyl-Dglucose.
When protoplasts at pH 5.5 were transferred from light to darkness,
"stored" light driving force maintained uptake in the dark at the full light
rate for the first 7 minutes. At pH 8, however, even 4 minutes after transfer
to dark, uptake was well below the light rate. Initial uptake rates over a
range of external concentrations were derived from progress curves obtained in the light and in the dark, both at pH 5.5 and at 7.7. When initial
rate was plotted against concentration, simple Michaelis-Menten kinetics
were observed only under the condition pH 5.5, light. In the dark at both
pH values, and in the light at pH 7.7, complex curves with intermediate
plateaus were obtained, strongly resembling curves reported for systems
where mixed negative and positive cooperativity is operating.
The same "K,. for protons" was observed in the dark and in the light
(10-7 molar). Switching protoplasts in the dark from pH 8 to 5.5 failed to
drive sugar transport by imposed protonmotive force, as judged by lack of
sensitivity to CCCP. Switching protoplasts which had taken up sugar in
the dark at pH 5.5 to pH 7 induced net efflux of sugar. Flux analysis
showed that this effect was entirely due to the prompt fall in influx.
It is concluded from the kinetic experinents that protonation alone is
not sufficient to convert the sugar transport system to its fiully activated
high affinity form. A further Light-dependent factor which acts synergisticafly with protonation is required.
The boundary membrane of a naked protoplast is in direct
contact with the solutes in the external medium, without the
intervening compartments of the cell wall and intercellular spaces
which are present in whole organs or tissue slices. These external
compartments have complicated flux measurements in higher
plant cells in the past, because initial flux should be estimated
from the intracellular content after brief influx periods; but most
of the substance taken up into a tissue after short-term incubation
is present in the extracellular compartments (2).
Use of protoplasts isolated from pea mesophyll cells recently
enabled us to measure initial flux of sugars through the plasmalemma in darkness and in light, and to detect within very few
minutes changes in flux which occurred as a result of transition
from light to darkness (8, 14). We reported that light uptake
differed from dark uptake in that it was sensitive to the proton
conductor CCCP2 but initially stable to various ATPase inhibitors
and to arsenate. Dark uptake from the start was sensitive to these
latter inhibitors but insensitive to CCCP. On transition from light
to dark at pH 5.5, the light driving force was apparently "stored"
for the first 7 min in the dark. During this period not only was
uptake at the accelerated light rate but it showed the inhibitor
sensitivity characteristic of light and not of dark conditions.
In the present communication, we report further on the characteristics of light and dark uptake in protoplasts, particularly in
relation to pH. It is currently widely believed (1, 3) that membrane
sugar transport involves co-transport of sugars and protons, driven
by the protonmotive force (the electrochemical potential gradient
for protons). According to a generally accepted model (e.g. 9),
protonation of a transport protein converts the sugar transport
system to a form with a high affinity for sugar, capable of uptake
from media of low sugar concentration. Among the findings we
present below is a demonstration, based on studies of initial
velocity versus concentration, that in our plasmalemma system
protonation alone is not a sufficient condition for conversion of
the system to its fully activated high affinity form. A further lightdependent factor is required, which acts synergistically with protonation.
MATERIALS AND METHODS
The plant material was Pisum sativum L. var. Dan. The methods
for growing and sampling the material, as well as for isolation of
protoplasts, were as described earlier (6, 8). Cellulysin concentration was 1%. In some experiments, the duration of Cellulysin
treatment was shortened from 20 h at room temperature to 2 h at
30 C; no effect of change in duration of Cellulysin treatment could
be detected on rate or kinetics of uptake.
Pretreatment and incubation took place in the modified Nagata
medium (6) buffered with Mes + Hepes (each 50 mM). Mannitol
concentration was 0.5 M. Retrieval of protoplasts from the incubation medium was carried out as described in (7) as was the
subsequent procedure for assaying radioactivity and internal standardization.
The light source for light treatments was as given in reference
8. Protoplasts to be incubated in the light were also pretreated for
l This research was supported by a grant from the United States-Israel
Binational Science Foundation (BSF), Jerusalem, Israel. The data are
taken from a dissertation to be submitted by M. G. to the Hebrew
2Abbreviations: CCCP, carbonyl cyanide
University of Jerusalem in partial fulfillment of the requirements for a
MeG, 3-0-methyl-D-glucose.
Ph.D. degree.
1146
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m-chlorophenylhydrazone;
Plant Physiol. Vol. 67,1981
ACTIVATION OF PROTOIPLAST SUGAR CARRIER
at least 1 h in the light; "dark" samples spent this period in
darkness.
The labeled sugars [14C]MeG, [3HJMeG, and ["4CJsucrose were
obtained from the Radiochemical Centre, Amersham, England.
RESULTS
Sensitivity to Light and to CCCP at Different External Proton
Concentrations. Figure la gives progress curves for MeG uptake,
in the light and the dark, at two pH values, during the "plasmalemma uptake phase" (8). It shows that the effect of light on MeG
uptake, previously reported when the pH of the medium was 5.5,
is also very marked at pH 8.
In addition Figure la shows the effect of the proton conductor
CCCP on MeG uptake in the light at the two pH values. CCCP
reduces uptake in the light to the dark level. The interesting fact
emerges that the sensitivity of uptake to pH remains undiminished
in the presence of the proton conductor, although presumably the
latter collapses the protonmotive force, either partly or completely.
The initial slope for uptake at pH 5.5 is about 3 times that at pH
8, both in the presence and absence of CCCP.
This result may also be expressed from a different view pointthe magnitude of the inhibitory effect of CCCP is no less at pH
5.5 than at pH 8, although in the latter case ApH across the
membrane has presumably been abolished or reversed.
CCCP was without appreciable effect on uptake in the dark,
either at pH 5.5 (8) or at pH 8.0 (not shown).
Sensitivity of Sucrose Uptake to Light, to pH, and to CCCP.
Sucrose is the "translocation sugar," the principal form in which
photosynthate is exported by mesophyll cells, and may pass in and
out of mesophyll cells en route to the vein. It was of interest to
check whether the membrane transport characteristics we observed
for glucose and other sugars (8) also applied to sucrose. Figure lb
demonstrates that sucrose uptake closely resembles MeG uptake
in its sensitivity to light and to pH. It is also sensitive to the proton
conductor in the light but not in the dark (not shown).
Effect of Light to Dark Transition at Acid and Basic pH Values.
If protoplasts in a medium at pH 5.5 are pretreated for 1 h in the
light, subsequent sugar uptake in the dark is at the accelerated
"light" rate for the first 7 min (8). Figure 2 shows an example of
an experiment to check whether this effect of "stored driving
force" is also evident at pH 8 (i.e. in the absence of a positive
A[H+] between medium and cytoplasm). At pH 5.5, uptake for
the first 7 min in the dark after light pretreatment again followed
the exact course of light uptake (Fig. 2). At pH 8.0, even 4 mi
after transfer from light to dark uptake was well below the light
rate. Nevertheless the rate of uptake by the light pretreated
protoplasts was somewhat above the dark rate.
Kinetics of Uptake (V versus S) in Dark and in Light at Two pH
Values. Figure 3a shows initial rate of uptake as a function of
external MeG concentration at pH 5.5 in the light and in the dark.
Figure 3b shows the same relationship for pH 7.7, again in the
light and in the dark. Each point in these figures has been obtained
from a time course of uptake at each concentration. Uptake was
10
pH 5.5 L
5
X
X
a
pH 55D
6
pH 5.5 L-D
0.
/,
0
12
1147
SO
7.5
TIME (min)
15
FIG. 2. Effect of light to dark transition on course of MeG uptake by
pea mesophyll protoplasts at two pH values. Light controls (0, A); dark
controls (0, A). Protoplasts transferred to dark at zero min, after 1 h
pretreatment in the light (O, A). MeG concentration I mm. Error bars
indicate + SD from plotted means of triplicates as in Figure 1.
CL
pH 8.0 L
~ ~ 7.5
E E
_L
15
pH 5.5 L
w
1b
' 2.5pH 5.50D
pH 800D
0
15
30
MINUTES
FIG. 1. Time course of uptake of MeG (a) and sucrose (b) by pea
mesophyll protoplasts at two pH values. In the light (0, O, A); in the dark
(@, E, A); in the light + 5 pM CCCP (O- - -1). Sugar concentration each
I mM. Error bars indicate ± SD from plotted means of three aliquots of the
protoplast suspension.
S (MeG concentration mM)
FIG. 3. Relation between initial velocity of MeG uptake by pea mesophyll protoplasts and external MeG concentration. a, at pH 5.5; b, at pH
7.7. In the light (0); in the dark (0). Initial velocity at each concentration
was derived from progress curves where uptake was followed over brief
time intervals (see text). Figures a and b taken from two separate experiments.
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GUY ET AL.
1148
assessed after 0.5, 1.5, 3.5, 6, and 9 min. The initial slope was
derived from the progress curves and is given in Figure 3. During
the 9 min, uptake was observed to be not far from linear.
Figure 3, a and b, shows that a curve close to a rectangular
hyperbola was obtained only under one set of conditions-pH 5.5,
in the light. This curve approximates to classic Michaelis-Menten
kinetics, as may be seen in the inset, where the plot V/S versus V
gives a reasonable straight line. Under all of the other conditions
shown in Figure 3, a and b, the curves obtained are not rectangular
hyperbolas; they are curves with intermediate plateaus, strongly
suggesting that cooperative effects are involved. They resemble
curves obtained for systems where mixed negative and positive
cooperativity is operating (10; see under "Discussion").
The "Km for Protons" in the Dark as Compared with the Light.
Dark uptake was earlier shown to be very different from light
uptake in sensitivity to inhibitors (8). The question arises as to
whether a totally different system mediates uptake in the dark, or
whether the same carrier system operates under both conditions
but is energized differently in light and in dark.
As an approach to this question we have studied the affinity of
the system for protons in the light and in the dark. Time curves
for uptake were obtained over the pH range 5 to 8 at intervals of
0.5 pH units. The initial rates of uptake were derived from these
time curves and are shown as a function of hydrogen ion concentration in Figure 4. Both curves approximate to rectangular hyperbolas. If a protonated carrier complex in fact plays a central
role in sugar transport, then the results shown in Figure 4 may
allow assessment of the Km of the carrier for protons (cf. 5, 9).
Linear transformation of the hyperbolas (see inset) indicate about
the same Km- 10-7 M, or pH 7, in both cases. (The slope of the
lines give 1/Km.) The intercept on the V axis (i.e. V,,a, rate of
uptake at saturating proton concentration) is, however, more than
4-fold higher in the light than in the dark.
Results obtained when the external MeG concentration was 10
mM, instead of I mM as in Figure 4, show a very similar pH
dependence (6, and unpublished data).
Attempt to Drive Sugar Uptake by Imposed pH Gradient in the
Dark. Our results are consistent with the notion (see under "Discussion") that in the light uptake may be driven by protonmotive
force. The CCCP stability of dark uptake, on the other hand,
suggests that this force is not operating in the dark. A possible
explanation might be that in the dark ApH across the membrane
is not maintained, and that ApH is the most influential component
of the protonmotive force at pH 5.5. We have attempted to impose
an artificial proton gradient across the plasmalemma in the dark
E
0~~~~~~~~~~~~
40_
.C
w
D
10
(
5
15
Sc Ht concentration, PM)
FIG. 4. Relation between initial velocity of MeG uptake by pea mesophyll protoplasts and proton concentration. In the light (0); in the dark
(@). Initial velocity at each pH value was derived from progress curves
where uptake was followed over brief time intervals. MeG concentration
I mM.
0
pH 5.5-pH
//
5.5,3
PH 5:5-pH 7.0o
Not flux pH4 5.5 -pH 7.0
7.0
....................... .
.
.~~~0
0~~~~~
.2~~~~~~~~~~~~~~~~~~
'5~~~~~~~~~~~~
6^/~pH
by first incubating protoplasts at relatively high pH (pH 8) then
switching them to low pH (pH 5.5). As criterion for the operation
of protonmotive force we have used sensitivity of sugar uptake to
the proton conductor CCCP. This technique has revealed protonmotive force driven sugar uptake by a similarly imposed ApH in
E. coli (4). In our case, measurements made even only 3 min after
the switch from pH 8 to pH 5.5 failed to detect CCCP sensitivity
of MeG uptake in the dark. Moreover, the reverse switch (incubation with MeG at pH 8 after pretreatment at pH 5) failed to
reveal a slower initial rate of uptake, as compared with protoplasts
pretreated at 8. A lower initial rate might have been expected as
a result of the imposed outwardly directed ApH. In all cases, the
rate of sugar uptake reflected the pH of the medium at time of
sugar supply, in accordance with Figure 4.
This last fact also emerges from the experiment shown in Figure
5 to be discussed below.
Effect on the Unidirectional Fluxes of Switching Protoplasts
from pH 5.5 to 7.0. In this experiment protoplasts were allowed to
take up ["4CJMeG for 50 min either at pH 5.5 or at pH 7.0. They
were then centrifuged briefly and resuspended in MeG medium
at the same concentration but now labeled with 3H. Aliquots of
protoplasts were removed at intervals and assayed for both 3H and
4C, allowing assessment of unidirectional influx and efflux, respectively. Half the protoplasts which had been at pH 5.5 were
switched to pH 7 at the time of transfer to 3H-labeled medium,
the other half continued at pH 5.5. The experiment was carried
out in the dark.
The following points emerge clearly from Figure 5 which presents the results:
1. Net influx was from the very start of the uptake period lower
at the higher pH.
2. Switching of protoplasts from pH 5.5 to 7.0 induced net
efflux of sugar. Sugar was thus lost from the protoplasts, even
though the bathing medium was at the same sugar concentration
as the previous one. This result strongly suggests that, even under
these dark conditions, an internal sugar concentration above the
external had been built up in some compartment of the cell during
incubation at pH 5.5.
E 20
2
Plant Physiol. Vol. 67, 1981
50
100
TIME (min)
FIG. 5. Progress curves for net MeG flux inwards (0) influx (1) and
efflux (E) into and out of mesophyll protoplasts. Latter were first incubated
for 50 min in 14C-labeled MeG (10 mM) either at pH 5.5 or at pH 7.0.
Those incubated at pH 5.5 were then transferred to MeG at same concentration but 3H-labeled, either at pH 5.5 or at pH 7.0. Efflux was followed
as loss of 14C from the protoplasts, influx as gain in 3H. Net efflux after
transfer from pH 5.5 to pH 7.0 (A).
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Plant Physiol. Vol. 67, 1981
1149
ACTIVATION OF PROTOPLAST SUGAR CARRIER
3. The net efflux of sugar after transfer to pH 7.0 was entirely result from the sum of two Michaelis functions. Summed Michaelis
due to the prompt response of influx, which fell quickly from the functions always give a smooth curve (e.g. 13). Curves with bumps
level which had characterized uptake at pH 5.5 to that which or intermediary plateaus are, however, known for a number of
characterized uptake at pH 7.0. A net loss of sugar after a similar enzymes (10, 20), and some of them bear a striking resemblance
pH switch has been observed for algae, and has been attributed to to the curves we show in our Figure 2 (20, Figs. lc and 8).
enhanced efflux (9), although in the latter case no flux analysis Koshland and his collaborators have studied the kinetics of such
was performed.
systems (10, 20). They concluded that the plateau indicates that
4. Unidirectional efflux was affected by the pH switch to only the binding protein possess at least three ligand binding sites, and
a slight degree. This result suggests that unidirectional flux is that the shape of curves of this type is due either to mixed
determined mainly by the H+ concentration on the same side (the negative-positive cooperativity in ligand binding, or to changes in
"cis" side) of the membrane. The "trans" H concentration (i.e. the the catalytic constants as a function of ligand (substrate) occuconcentration on the opposite side) had only a small effect on pancy.
efflux, but it was consistent in repeated experiments-efflux was
Further, Koshland (11) has also pointed out that if a system has
somewhat depressed by the switch to lower trans H+ concentration. been converted to the fully active form before the addition of
DISCUSSION
Evidence we have collected here and in an earlier communication (8) would be consistent with the current hypothesis that the
driving force for membrane sugar transport is the electrochemical
potential gradient for protons (AjH+), but only in the light. In
particular, the "stored" driving force evident during the first few
minutes after transition of protoplasts from light to darkness at
pH 5.5 is most reasonably explained as residual AjH+. This follows
from a consideration of its stability or sensitivity respectively to a
variety of inhibitors (8). The question arises from the present
study as to why the stored driving force is capable of maintaining
initial dark uptake at the full light rate if the protoplasts are at pH
5.5, which this is not the case at pH 8.0. We offer the following
possible solution to this question:
At pH 5.5, the electric potential component of AAH+, A, iS
much less influential on sugar uptake than is ApH. This has been
observed for Chlorella by Schwab and Komor (16); and has also
been reported by Kaback and his collaborators (see 12) for E. coli
membrane vesicles with regard to a considerable number of
transport systems including that of glucose-6-P. At pH 8, on the
other hand, in the absence of an inwardly directed ApH, uptake
is sensitive to A+. This has been observed both for Chlorella (16)
and for the E. coli system (12). Light may greatly stimulate the
plasmalemma-sited proton extrusion pump. This has been shown
for Nitella by Spanswick (17, 18) and for oat protoplasts by
Rubinstein and Tattar (15); and has also been proposed by van
Bel (21) for tomato internode discs. (In our system, the pump may
scarcely operate in the dark, as judged from the observation that
fusicoccin only stimulates sugar uptake in the light [Guy et al., in
preparation].) On transfer to darkness, loss of electrogenic pump
activity may result in a drop of potential (noted by Spanswick,
17), the half time for decay being about 0.5 min (23, p. 246). The
driving force for sugar uptake at pH 8 may thus have diminished
too fast in our protoplasts on transfer to darkness for us to observe
it. We deduce that the ApH created by the pump, which is the
principal driving force at pH 5.5, decays far more slowly and thus
allows uptake at the accelerated light rate for some minutes after
transition to dark.
A striking finding in the present investigation is that classic
Michaelis-Menten kinetics are observed when uptake is taking
place at pH 5.5 in the light, but that in the dark, or at high pH in
both light and dark, the curves relating initial uptake velocity to
external MeG concentration are multiphasic and show intermediary plateaus (Fig. 3), suggesting complex cooperativity (see
below). It has been suggested for Chlorella (9, 19) as for many
systems that the glucose carrier exists in two different states for
binding sugar, a protonated form with a high affinity for sugar,
and an unprotonated low affinity form. Between approximately
pH 6.5 and 8.5, in the case of Chlorella, it is claimed that a mixture
of the two forms is present and that total uptake is the result of
the joint operation of both forms of carrier. A disadvantage of
this proposal, however, is that curves with bumps (cf. 19) cannot
substrate, then the subsequent binding of substrate will proceed
without further conformational change and will follow a simple
Michaelis-Menten pattern. On the basis of the contrasting curves
we show in Figure 3, we suggest that under the condition of pH
5.5 light, the transport system is present in its fully active form, as
indicated by the Michaelis-Menten kinetics. Protonation alone is
not sufficient to convert it to this fully active form, since in the
dark the intermediary plateau type of curve was obtained even
when the medium was at pH 5.5. (The system was indeed protonated in the dark at pH 5.5, as is indicated by the analysis of rate
of uptake versus proton concentration shown in Fig. 4.) We
conclude that some other factor is operating in the light, which,
together with protonation, brings about full activation. This other
factor, might be the electric field imposed by a large membrane
potential, which may only be achieved in the light (17, 18). It
could also be some other factor differentiating an illuminated
from a nonilluminated protoplast. In the absence of the light
factor, ligand energy eventually brings about full activation of the
system, as evidenced by the fact that, with increasing substrate
concentration, the dark curve approaches that obtained in the
light (Fig. 3, a and b).
CCCP reduces light uptake almost exactly to the dark level
(Fig. la). This finding, we suggest, is not fortuitous. Under both
conditions, uptake is proceeding unaided by the proton extrusion
pump-in the former case because the latter is short-circuited by
the proton conductor, and in the latter case because it may require
light to activate the pump in our cells (cf. 15, 17, 18, 21). Yet dark
uptake does not appear to be facilitated diffusion. The ratio of
cytoplasmic to external concentration after 15 min of uptake
(assuming cytoplasmic volume to be 5% oftotal protoplast volume)
has frequently exceeded 5 when external concentration is below
1 mM. The net efflux of sugar shown in Figure 5 when protoplasts
were transferred from pH 5.5 to pH 7 strongly suggests that
accumulation above the external concentration had occurred at
pH 5 in some cellular compartment.
Dark uptake was highly sensitive to pH, as is demonstrated by
the immediate response of the unidirectional fluxes to changes in
cis-pH (Fig. 5) as well as by the relationship shown in Figure 4.
Further, light uptake remained pH sensitive in the presence of
CCCP (Fig. 1). The apparent conflict between this latter finding
and that of van Bel (21) is probably resolved by the fact that initial
flux was observed in our investigation whereas his measurements
were made after some hours. This strict pH dependence even in
the presence of CCCP, together with the very similar pH dependence of vacuolar uptake although the direction of the protonmotive force is here reversed (14) are strong indications that the pH
sensitivity may reflect a requirement of a constituent of the
transport system for protonation and is not necessarily reflecting
the energy input for transport. We have previously suggested, both
in connection with dark uptake (8) and with uptake by vacuoles
(7) that direct fueling by ATP may be one of the possible modes
of energization of sugar transport in protoplasts
as
it is in bacteria
(22). Whether dark uptake and hght uptake are mediated by the
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GUY ET AL.
1150
same system differently energized, as the pH sensitivity might
suggest, or whether totally different systems are involved, must
await further clarification.
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