Jun 25, 1987 - WILLIAM J. LUCAS* AND CLYDE WILSON. Botany Department, University ofCalifornia, Davis, California 95616. ABSTRACr. Experiments were ...
Plant Physiol. (1987) 85, 423-429 0032-0889/87/85/0423/07/$0 1.00/0
Influence of Mannose on the Apoplasmic Retrieval Systems of Source Leaves' Received for publication May 8, 1987 and in revised form June 25, 1987
WILLIAM J. LUCAS* AND CLYDE WILSON Botany Department, University ofCalifornia, Davis, California 95616 only slowly metabolized (10, 16, 23). Since the transport of Pi between the vacuole and cytosol is thought to be slow (23, 27; Experiments were conducted in which -mannose was supplied to but see Ref. 16), the addition of mannose effects a sequestration mature Beta vulgaris L. (sugar beet) leaves, via the transpiration stream, of cytosolic Pi (5, 13; but cf Ref. 16). Inhibition of sugar transport to perturb photosynthetic carbon allocation by sequestering cytosolic Pi. by mannose has been previously documented, e.g. glucose uptake Biochemical and enzymic analyses conducted on this tissue indicated that into (15) and sucrose storage in corn scutellum (9) are both nannose 6-P was present, that it was only slowly metabolized, and that inhibited by mannose treatment. The work conducted on corn after a 24-hour pretreatment sugar metabolism was slightly perturbed. scutellum indicated that mannose treatment reduced the ATP However, sucrose retrieval by the mesophyll tissue was greatly impaired level in this tissue, and Humphreys postulated that this may have in 24-hour mannose-pretreated tissue, a response which was due in part a significant role in the inhibitory response of the tissue to to mannose acting as an osmoticum. Inhibition of glucose, fructose, and mannose (see also [28]). arginine uptake into mannose-treated sugar beet leaf discs indicated that In the present study, we further investigated the effect of mannose may elicit a general perturbation of all membrane transport mannose on the transport of sucrose, glucose, fructose, and processes. This conclusion was supported by our frmding that sucrose arginine into mature leaftissue of sugar beet. Our results indicate efflux was increased from mannose-treated tissue. Analysis of adenine that the inhibition of plasmalemma transport is not limited to nucleotide levels showed that whereas these levels declined over the first sucrose as the influx of these other substrates was also reduced. 3 to 6 hours of the mannose treatment, by 24 hours they had recovered Furthermore, the inhibitory aspects of mannose cannot be exto near control values. Similar experiments conducted on Nicotiana plained by a reduction in adenine nucleotide levels alone since, rustica indicated that whereas mannose 6-P was present in mature leaves, after a 24 h mannose pretreatment, these levels recovered to it remained at a much lower level than that found in sugar beet. Sucrose near-control values. Since tobacco has been reported to be able uptake into N. rustica was insensitive to mannose pretreatment. However, to metabolize exogenously supplied mannose (5, 12, 13, 20), we glucosamine treatment, which is also thought to sequester cytosolic Pi, selected two species of Nicotiana, N. rustica and N. tabacum, to inhibited sucrose uptake in both N. rustica and B. vulgaris. Further, form a comparative basis for this study. We found that whereas experiments conducted on N. tabacum L. var Xanthii showed that man- sucrose influx into N. rustica was completely insensitive to nose caused an inhibition of sucrose uptake, indicating that a range of mannose pretreatment, transport into N. tabacum was inhibited. sensitivity to mannose exists between closely related species. These Glucosamine pretreatment inhibited sucrose influx into N. rusresults are discussed in terms of possible mechanisms of inhibition. tica and sugar beet. Collectively, these studies implicate a role of cytosolic Pi in the regulation of transport across the plasmalemma. ABSTRACr
In a recent study on the regulation of sucrose transport across the plasmalemma of sugar beet mesophyll cells, we used mannose to alter the partitioning of carbohydrates between the cytosol and the chloroplast. In sugar beet, mannose did not reduce the sucrose level in the manner expected from reports in the literature (5, 10, 13, 14); however, it did cause a reduction in exogenous ['4C]sucrose influx. In addition, mannose pretreatment prevented, in some way, the change in partitioning of exogenously supplied ['4C]sucrose, which was found to occur following the removal of sink activity (31). These findings suggested that further studies, using mannose as an experimental treatment, may provide information on both the regulatory mechanisms involved in transport at the plasma membrane and carbohydrate allocation in relation to 'phloem loading.' It is generally accepted that in species lacking mannose isomerase, mannose is able to reduce cytosolic inorganic phosphate (Pi) levels by phosphorylation to mannose 6-P, which is then '
Supported by National Science Foundation Grant PCM 83-15408 to
W. J. L.
423
MATERIALS AND METHODS Plant Material. Beta vulgaris L. (var SSBN1, USH10, Lot 3102 or USHl1, Lot 82313) seeds were planted in a mixture containing vermiculite, peat moss, and sand (2:1:1 by volume) and grown in a controlled-environment chamber under a 27°C day/17°C night temperature regime and a 16 h photoperiod (250 gmol m'1 s-'). The plants were watered daily and fertilized twice weekly with Hoagland solution. The third pair of leaves (after the cotyledons) of 4-week-old sugar beet plants were used. Nicotiana tabacum L. var xanthii and Nicotiana rustica L., were planted in the soil mixture described above and grown under greenhouse conditions. Plants were fertilized during the third, fifth, and seventh weeks with ammonium phosphate. Experimental plants were 9 weeks old. Kinetic Studies. Kinetic uptake studies on B. vulgaris were performed using the method of Maynard and Lucas (21) as modified by Wilson et al. (32) and Wilson and Lucas (31). Radioactive sugars (D_[U-_4C]sucrose, -fructose, and -glucose) were obtained from International Chemical and Nuclear Corp. (Irvine, CA). Radioactive arginine (L-[U-3H]arginine) was obtained from NEN Research Products (Boston, MA). The specific activities of the uptake solutions for all sugar and arginine
424
LUCAS AND WILSON
experiments were 1.78 x 107 Bq mmol-'. In Nicotiana experiments, a segment of the mature tobacco leaf was excised and placed on a rubber pad located in a dish containing a solution of 20 mm CaCl2 and 60 mM Mes buffer (pH 5.0). Leaf discs, 4 mm in diameter, were cut and then pretreated for 30 min on a solution containing 20 mm CaCl2, 120 mm Mes (pH 5.0), and enough mannitol to adjust the total osmolality to 200 mOsm; sugars at the concentration specified for the particular experiment were also included. This pretreatment was followed by a 30-min uptake period, which was terminated by using three 7min washes in media containing 20 mm CaCl2, 60 mm Mes (pH 5.0), and the appropriate sugar at 40 mm. Radioactivity within the sugar beet and tobacco leaf discs was quantitated using a Beckman 9800 scintillation spectrometer (Beckman Instruments Inc.) as previously described (22).
ATP Extraction and Determination. Leaf discs that had been treated using the same protocol employed for kinetic experiments were quickly frozen using liquid N2. These discs were then lyophilized for 18 h in a DURADRY freeze-dryer equipped with a bulk tray dryer (FTS Systems, Stone Ridge, NY). Lyophilized samples were weighed and then ground, using a ground glass homogenizer, in 2.0 ml of ice-cold 1.0 M HC104; 2 mg ml-' of Polyclar AT (Polysciences, Warrington, PA) was also added to the extraction medium (18). The homogenizer was rinsed with 1.0 ml of 0.05 M phosphate buffer (pH 7.5), and after allowing 20 min for complete extraction and protein denaturation, the rinse and the extract were combined. The mixture was then centrifuged at 25,000g for 30 min at 4°C. The supernatant was neutralized to pH 7.5 with 5 N KOH. After waiting 20 min to allow for the precipitation of potassium perchlorate, the extract was again centrifuged for 30 min at 25,000g. The supernatant was stored at -70°C until assayed (2-3 d). A modification of the procedures of Yoshioka and Tamura (33) and Preston (26) was employed to determine AMP, ADP, and ATP levels (32). Chromatography was performed using a Beckman Series 344 Gradient Liquid Chromatograph equipped with an Ultrasphere IP column (4.6 mm diameter, 250 mm long). The eluting peaks of the adenine nucleotide 1,N6-ethenoadenosine-derivatives (32) were detected using a fluorescence detector (Beckman model 157) and analyzed on an Altex Chromatopac C-R I B data processor (Beckman Instruments). Peaks were identified by using retention times of standards. Further confirmation of the peaks in the extracts was obtained enzymically. Addition of pyruvate kinase and adenylate kinase (Sigma) or pyruvate kinase alone, to extracts, resulted in the disappearance of the peaks which were identified as AMP and ADP, respectively, while the ATP peak increased. Addition of hexokinase or calcium phosphate to the extract resulted in the loss of the putative ATP peak. Sugar, Starch and Mannose 6-P Analysis. Sugars and starch were extracted from leaf tissues and analyzed using the procedures and protocols described by Wilson and Lucas (31). Mannose 6-P was extracted according to the procedure of Herold et al. (14). The extraction procedure was similar to our sugar protocol (31), except that 20 leaf discs were used and the ethanol extractions were followed by two extractions using hot water. The extracts were evaporated to dryness under a stream of nitrogen gas and stored at -70°C until assayed (1-2 d). To assay the extracts for mannose 6-P, the samples were first rehydrated to 0.5 ml, and 50 ,l of this sample was added to a 13 x 100 mm culture tube containing 2.82 ml of 50 mm HepesNaOH buffer (pH 7.6), 100 ,l of NADP (12 mM), and 10 ,l each of glucose 6-P dehydrogenase (G6PDH, 340 units ml-'), phosphoglucose isomerase (PGI, 580 units ml-'), and phosphomannose isomerase (PMI, 140 units mlr'). This mixture was incubated for 1 h by which time the stoichiometric production of 1 NADPH for each mannose 6-P molecule was complete. The
Plant Physiol. Vol. 85, 1987
resulting NADPH was measured spectrophotometrically at 366 nm against a blank containing 10 ,l distilled water in place of phosphomannose isomerase. (G6PDH from Bakers yeast, PGI from Bakers yeast type 1II, and PMI from yeast along with NADP were obtained from Sigma.) Mannose and Glucosamine Pretreatments. Three h into the photoperiod, the leaf-petiole complex of the third pair of leaves (after the cotyledons) were excised from the base of the crown while the sugar beet plant was submerged underwater. The leafpetiole complex was then either placed in distilled water (control) or a 10 mM mannose (or glucosamine) solution before being returned to the controlled-environment chamber. After predetermined intervals, leaf discs (4 mm diameter) were punched from the leaf and used for sugar analyses and kinetic experiments (same protocol was followed for tobacco plants). When appropriate, 10 mm mannitol (or sorbitol) was substituted for mannose. ['4CiSucrose Efflux Experiments. As in sucrose uptake experiments, the third pair of leaves (after the cotyledons) from 4week-old sugar beet plants were used in our efflux studies. Leaves were cut from the plant and submerged in a 20 mm CaCl2 solution while the lower epidermis was peeled away. Leaf discs were excised and then pretreated, for 30 min, on a medium containing 20 mm mM sucrose, 120 mm Mes (pH 5.0), and mannitol added to adjust the osmolality to 500 mOsm. Discs were then transferred to an identical medium containing 20 mM ['4C]sucrose. After a 30-min loading period, the discs were transferred to fresh pretreatment medium. At predetermined times, discs were again transferred to fresh media, and aliquots of the efflux solution were analyzed for radioactivity by liquid scintillation counting. At the end of the experiment, the discs were analyzed for residual radioactivity. Efflux data were analyzed according to Pitman (25) and Cram (6), the results being interpreted in terms of an apoplasmic and two intracellular compartments (vacuole and cytosol) arranged in series. In certain instances some of the leaf discs were analyzed for ethanol-soluble sugars as previously described. Radioactivity remaining after ethanol extraction followed by cold water extraction was attributed to starch. RESULTS Inhibitory Effect of Mannose on Sugar and Amino Acid Transport. Wilson and Lucas (31) recently showed that pretreatment of sugar beet leaves with mannose results in a progressive decline in the capacity of these leaves to retrieve exogenously supplied ['4C]sucrose. This inhibition by mannose may have been due to its osmotic rather than biochemical properties; this possibility was tested by substituting mannitol (or sorbitol) for mannose. After a 24-h mannitol treatment, the capacity of the leaves to take up ['4C]sucrose was slightly decreased, whereas a similar treatment with mannose caused a strong inhibition of sucrose influx (Fig. 1). This suggests that mannose has effects independent of its osmotic properties. (An equivalent inhibition was obtained with either sorbitol or mannitol.) Since mannose had dramatic effects on sucrose influx, apart from a small osmotic effect, we decided to examine its influence on the transport of other sugars and amino acids. As in the case of sucrose, mannose pretreatment resulted in a decrease in the transport of both ['4C]fructose (Fig. 2A) and [(4C]glucose (Fig. 2B). However, in contrast with sucrose, the uptake of these sugars, after mannose pretreatment, was mediated solely by the first-order kinetic component. We extended our investigation on the specificity of the mannose effect by measuring the influence of mannose on amino acid transport. Dual-label ([3H]arginine and [14C]sucrose) experiments were performed in these studies. Control experiments showed that arginine was taken up via the combined effect of a saturable and a first-order kinetic system (see also Ref. 22), with
425
INHIBITION OF SUCROSE UPTAKE 20
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ructose
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FIG. 1. Effect of mannose and mannitol on net sucrose influx into B. vulgaris leaf discs. Petioles were excised at the base, while the crown of the plant was submerged underwater. The petioles were then placed in either distilled water (0), 10 mm mannitol (0), or 10 mM mannose (A) before being returned to the controlled-environment chamber. After 24 h, kinetic studies were performed. A 30-min preincubation period in unlabeled sucrose was followed by a 30-min incubation in ['4C]sucrose. Uptake solutions were adjusted to 500 mOsm with mannitol. Each point represents the mean ± SE for two sets of six replicates.
15
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the major contribution occurring via the first-order kinetic system. As with sugar influx, mannose pretreatment resulted in an inhibition of both the first-order and saturable components of [3H]arginine uptake (Fig. 3). Hence, the inhibitory influence of mannose appears to be related to either a general cellular- or membrane-related phenomenon. Adenine Nucleotide Levels Affected by Mannose Pretreatment. It has been suggested that mannose may reduce sugar transport into cells through its sequestration of cytosolic Pi. This removal of Pi would reduce the level of ATP available for membrane transport. To investigate this hypothesis, we analyzed the levels of adenine nucleotides in mannose-treated and control tissue over a 24-h period (Table I). We found that leaves which had been pretreated with mannose for 3 h contained lower levels of AMP, ADP, and ATP compared with control leaves. However, after 24 h, the adenine nucleotide levels in the mannose-treated leaves had recovered to near-control values while, during this period, membrane transport activity continued to decline. It would appear from these data that mannose is acting by some other means to inhibit membrane transport. Mannose 6-P Levels in Maznose-Pretreated Leaves. It is possible that the mannose-induced inhibition of membrane transport is related to either the sequestration of cytosolic Pi or the subsequent formation of mannose 6-P, per se. To examine this possibility, we sampled control and mannose-treated leaves at various intervals over a 24-h experimental period and determined the mannose 6-P levels. We were unable to detect mannose 6-P in control leaves, but in mannose-treated leaves, the mannose 6-P levels progressively increased throughout the light period and then decreased during the subsequent dark period (18 h) (Table II). During the following light period, the mannose 6-P level returned to the pre-dark value. These data indicate that Beta vulgaris is capable of metabolizing mannose 6-P, although the rate appears to be too low to prevent its accumulation in this tissue. Effect of Mannose on Nicotiana. Having characterized the
^
|
Glucose
Mannose
Treated
0 0
z
20
10
Sugar
30
40
(mM)
Concentration
FIG. 2. Effect of mannose pretreatment on net fructose (A) and net glucose (B) influx into B. vulgaris leaf discs. Experimental techniques were as
Figure 1. Points for both the fructose control (A), (A), and glucose control (0), and mannose-treated (U)
described in
mannose-treated
represent the
means ± sE
for two -sets of six replicates. Sucrose control
(0) is included for reference. effect of mannose
species,
on
sugar
N. rustica and N.
has been
reported
uptake in B. vulgaris, we selected two tabacum, for study, since Nicotiana
to be mannose-tolerant.
beet, transport into N. rustica leaf unaffected by mannose pretreatment (Fig. 4A). In order to determine if affected leaf carbohydrate metabolism, In
discs
contrast
to sugar
sucrose
was
mannose
we
analyzed the
starch and
sugar levels within the tissue.
As
found in our studies with sugar beet (31), mannose-pretreat(ment caused the starch levels to decrease, while sugar levels increased slightly (Table III). We also measured the mannose 6-P levels in N.
rustica leaves after 24-h control and
(Table II). Although
we
treatments
mannose
could detect the presence of
mannose
P in leaves of N. rustica treated with mannose, the levels
lower
than
those found
in B. vudgaris after
3 h
of
6-
were
mannose
treatment. Either N. rusuica is capable of metabolizing mannose 6-P more quickly than sugar beet, resulting in lower mannose 6P
levels,
or
the lower levels of
of mannose into tobacco
mannose
is supported by slower uptake of these data indicate
chemical
sucrose
that in N. rustica,
responses, but that magnitude to perturb-or are
these
on
(Fig. 4A).
Collectively,
mannose can
are
tolerated
transport system of this plant. We furthered investigations our
6-P reflect slower uptake
mesophysl tissue. The second possibility elicit bio-
either insufficient more
tobacco
in
by-the sucrose
by examining the
426
LUCAS AND WILSON
levels in !tr. rustica and thereby elicit a decrease in sucrose influx. Herold et al. (13) have suggested that glucosamine sequesters Pi in tobacco leaves. Glucosamine pretreatment of N. rustica leaves resulted in an inhibition of both the saturable and linear components of uptake, with the saturable system being the most affected (Fig. 5). However, the extent of glucosamine-induced inhibition was not as severe as that observed with sugar beet (Fig. 5). Effect of Mannose on Sucrose Efflux. Herold (11) suggested that mannose had marked effects on the membrane permeability of spinach beet leaf tissue. We performed efflux studies in order to determine if sucrose efflux was also affected in sugar beet. The effect of a 24-h mannose pretreatment on ['4C]sucrose efflux is presented in Fig. 6. In control tissue the half-time for ['4C]sucrose exchange across the plasmalemma was 40.5 min, whereas in mannose-treated tissue this half-time was reduced to 18.8 min. Mannose also affected the distribution of ['4C]sucrose in the cytosolic and vacuolar compartments. In control leaves, only 5% of the ['4C]sucrose was present in the cytosol, while in mannosetreated tissue this value increased to about 25%. Thus, it would appear that mannose can affect intracellular transport processes in addition to perturbing the fluxes crossing the plasmalemma.
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5
0 0 z
Plant Physiol. Vol. 85, 1987
10 20 30 40 Arginine & Sucrose Concentration (mM)
FIG. 3. Effect of mannose pretreatment on net [3H]arginine influx into B. vulgaris leaf discs. Experimental techniques were as described in Figure 1. For kinetic studies, the preincubation media contained the appropriate concentrations of D-sucrose and L-arginine, while uptake solutions contained both ['4C]sucrose and [3H]arginine. The points for the arginine and sucrose controls (A, 0), respectively, and for net arginine influx and net sucrose influx into mannose-treated leaves (A, 0), respectively, represent the means ± SE for two sets of six replicates.
effect of mannose on another species, N. tabacum. Our results with this species were unexpected. A 24-h mannose pretreatment resulted in a dramatic decrease in both the first-order and saturable components of sucrose influx (Fig. 4B). This mannose response is similar to that obtained with B. vulgaris and indicates that there may be subtle differences among tobacco species, with respect to carbohydrate biochemistry. Since sucrose influx was also greater in N. tabacum, as compared with N. rustica, we cannot exclude the possibility that a difference in mannose transport is the basis for this discrepancy in species sensitivity to mannose.
Effect of Glucosamine on Sucrose Influx. If the sequestration of Pi is involved in the inhibition phenomenon, it should be possible to experimentally effect a reduction in the cytosolic Pi
DISCUSSION In the present study, we used mannose and glucosamine to probe the regulatory processes involved in sugar transport into mature leaf tissues of sugar beet and tobacco. Mannose has been used in various plant systems as a tool to investigate the role of cytosolic Pi in regulating various aspects of photosynthetic carbon metabolism (8, 10, 13, 30). It is currently accepted that mannose sequesters cytosolic Pi through its reaction with Pi to produce mannose 6-P, via hexokinase. Many plant tissues either lack phosphomannoisomerase, or this enzyme is compartmentalized such that mannose 6-P is metabolized very slowly (10, 23). In our experiments, mannose pretreatment caused a dramatic perturbation of sugar influx into mesophyll tissue of sugar beet plants. Mannose pretreatment resulted in a progressive decrease in sucrose uptake over a 24-h period (31). Further studies revealed that uptake of both glucose and fructose was also reduced, indicating that the mannose effect may be a consequence of a more general phenomenon. This interpretation is supported by the observation that arginine uptake was similarly affected. Consistent with this view is the observation that mannose also influenced sucrose efflux from sugar beet leaf discs. Our experi'ients indicate that both the plasmalemma and tonoplast are Sfected by mannose, as the half-times for sucrose efflux from both cytosolic and vacuolar compartments were decreased relative to control values. Although some caution is needed in the interpretation of the efflux data obtained in this manner (4), we
Table I. Adenine Nucleotide Levels in Mannose-Treated Sugar Beet Leaves Leaves were excised at the base of the petioles while the crown of the plants was submerged underwater. The petioles were either placed in 10 mM mannose or distilled water (control) before being returned to the controlled-environment chamber for the times specified. Adenine nucleotide levels were determined by HPLC. Data are from two experiments. AMP ADP ATP Treatment Duration Total pmol mg' dry weight h 259 419 802 1471 0 None 176 251 349 Mannose 3 776 314 329 Control 832 1475 235 Mannose 223 368 6 826 294 350 Control 839 1483 677 386 373 1416 24 Mannose 269 302 736 Control 1306
427
INHIBITION OF SUCROSE UPTAKE Table II. Mannose 6-Phosphate Levels in Mannose-Treated Sugar Beet and Tobacco Leaves Leaves were excised at the base of the petioles while the crown of the plants was submerged underwater. The petioles were either placed in 10 mm mannose or distilled water (control) before being returned to the controlled-environment chamber for the times specified. Experimental treatments were started 3 h into the photoperiod; the controlled-environment chamber was programmed for a 16-h light:8-h dark cycle. Mannose 6-P levels were determined by enzymic assay. Mannose 6-P Plant Time Control Mannose-treated h utmol mg-' Chl ± SE NDa ND 0 Sugar beet 3 ND 1.3 ± 0.1 6 ND 1.8 ± 0.1 ND 9 1.8 ± 0.3 ND 12 2.0 ± 0.3 ND 18 1.1 ± 0.1 24 ND 2.3 ± 0.2 ND ND 0 Tobacco (N. rustica) ND 0.7 ± 0.1 24 a Not detected.
feel our interpretation of the data as representing efflux from a three-compartment model is justified (at least to a first approximation) by a comparison of plots of log Qt and log Qt/dt versus time as suggested by Cram and Laties (7) and further emphasized by Kochian and Lucas (17). By extrapolating the efflux curves to t = 0, we were able to estimate the relative amounts of radiolabel present in the vacuolar and cytosolic compartments. At the end of a 30-min uptake period only 5% of the label resided in the 'cytosolic compartment' of sugar beet source leaves. This result is similar to the findings of Thom et al. (29) for their study of efflux of sugars from cultured sugar cane suspension cells. They reported that while most of the label was in the cytoplasm following the initial minutes of uptake, the majority of label was subsequently located in the vacuole. However, after mannose pretreatment, 25% of the ['4C]sucrose was located in the cytosolic compartment of the sugar beet leaf. Evidently, mannose may either directly or indirectly affect the partitioning of sucrose between the vacuole and the cytosol. An important aspect of the mannose effect on sugar uptake was the difference in the pattern of inhibition displayed by sucrose, glucose, and fructose. Although uptake of sucrose was reduced by mannose pretreatment, the resulting kinetic profile demonstrated that influx was still mediated by both the firstorder and saturable components. However, for fructose and glucose, mannose pretreatment completely inhibited the saturable transport component; influx was mediated solely by the firstorder component. Perhaps there exists a basic difference in the regulation of the various sugar-transport systems. Previous investigators have documented reduced synthesis of ATP upon the addition of mannose and have used this finding as a basis to explain their observations (9, 28). In the present study, we found that mannose addition reduced adenine nucleotide levels when measured after 3 h and that this correlated with decreased sugar influx. However, after 24 h, adenine nucleotide levels recovered to near control values, while sugar transport activity continued to decline. Based on this finding, we feel the explanation for our observed mannose response must lie elsewhere. An interesting outcome of this study was the discovery that two closely related species of Nicotiana differed widely in their
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10
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c
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10
5
0 0
10
20
30
40
Sucrose Concontration (mM)
FIG. 4. Effect of mannose pretreatment on net ['4C]sucrose influx into N. rustica (A) and N. tabacum var xanthii (B) leafdiscs. Experimental techniques were as described in Figure 1, except that the osmolality of the solution was adjusted to 200 mOsm. The points for the controls (0, 0) and mannose-treated discs (0, *) represent the means ± SE for two sets of six replicates.
response to mannose. N. rustica seemed very tolerant to mannose pretreatment even after 24 h, while N. tabacum var xanthii reacted in a manner similar to that observed with sugar beet. Differences in phosphomannoisomerase activity or compartmentalization might account for the observed differences. Although N. rustica appeared to be tolerant of mannose pretreat. ment, we were able to inhibit sucrose influx by substituting glucosamine for mannose. Since glucosamine is reported to sequester Pi in tobacco, our results imply that changing cytosolic Pi levels may be important in the regulation of sugar transport across the plasmalemma in tobacco as well as sugar beet. On a speculative note, it may be that both mannose and glucosamine elicit a general perturbation of membrane transport by interrupting a phosphate-dependent component of the phosphatidylinositide cycle (1). Support for this hypothesis can be found in the plant literature, insofar as data is now available that the phosphatidylinositide cycle is present in plant cells (2, 24). Further, lithium, an inhibitor of phosphatidylinositide reincorporation into the plasma membrane (1), also inhibits sugar transport. Carlier and Thellier (3) reported that lithium treatment
428
LUCAS AND WILSON
Plant Physiol. Vol. 85, 1987
Table III. Influence of Mannose and Glucosamine on Sugar and Starch Content in Mature Leaves ofN. rustica Leaf petioles were excised at the base while the crown of the plant was submerged underwater. The leafpetiole complex was either placed in 10 mM glucosamine or 10 mm mannose when appropriate or distilled water (control) and then returned to the controlled-environment chamber for 24 h. Sugar Content Treatment Starch Sucrose Glucose Fructose
,tmol glucose
,smol mg-' Chi ± SE Control (untreated) Mannose-treated Control Glucosamine-treated
4.9 ± 0.3 6.0 ± 0.6 5.4 ± 0.2 10.5 ± 0.9
4.3 ± 0.3 5.6 ± 0.4 2.5 ± 0.3 6.5 ± 0.5
equivalents
mg1' Chl ± SE 0.9 ± 0.1 1.5 ± 0.3 0.8 ± 0.1 6.9 ± 0.5
5.270
127.6 ± 11.8 87.1 ± 7.1 547 ±41 410 ± 46
Control
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40
FIG. 5. Effect of glucosamine pretreatment on net ['4C]sucrose influx into N. rustica (A) and B. vulgaris (0) leafdiscs. Experimental techniques were as described in Figure 1, except 10 mm glucosamine was substituted for mannose. The points for controls (A, 0) and glucosamine-treated (A, 0) leaves represent the means + SE for six replicates.
prevented the induction of methyl-glucose transport into Pelargonium leaf discs. Application of lithium to discs that had undergone transport induction caused a large reduction in transport capability. In Ipomoea, the lithium inhibition of sucrose retrieval can be reversed by the addition of myo-inositol (MA Madore, personal communication). Future work will investigate the possible involvement of the phosphatidylinositide cycle in the regulation of sugar (sucrose) retrieval systems in mature mesophyll tissue, as well as the related phenomenon of wound recovery (19). Acatowledgments-We wish to thank Drs. J. Cronshaw, M. A. Madore, and C. Ralph Stocking for their valuable advice and discussion throughout the performance of thit research, and to Lyn Noah for typing the manuscript.
1
2
3 4 Time (h)
5
6
7
FIG. 6. Kinetics of ['4C]sucrose efflux from control (A) and mannosetreated (B) leaf discs of B. vulgaris. Leaf discs were loaded for 30 min in 20 mm ['4C]sucrose. The efflux solution contained 20 mm sucrose, 120 mM Mes (pH 5.0) and enough mannitol to achieve 500 mOsm. The data are plotted as the logarithm of cpm remaining in the tissue (Q,) versus time. The slowest effluxing component (vacuole) was extrapolated to zero and subtracted from the overall curve. The insets represent cytoplasmic efflux from control (A) and mannose-treated (B) tissues. The half-times for exchange of the cytoplasmic compartment was 40.5 and 18.5 min for control and mannose-treated tissue, respectively. LITERATURE CITED 1. BERRIDGE MJ, RF IRVINE 1984 Inositol triphosphate, a novel second messenger in cellular sgal transduction. Nature 312: 315-321 2. Boss WF, MO MASSEL 1985 Polyphosphoinositides are present in plant tissue culture cells. Biochem Biophys Res Commun 132: 1018-1023 3. CARLIER G, M THELLIER 1979 Lithium-perturbation of the induction of a
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