- Email: [email protected]
Sucrose Uptake in the Xylem of Populus
Sh ort Com m unication
Sucrose Uptake i n the Xylem of Populus JORG J. SAUTER
Botan ismes Institut del' Universitat Kiel, OlshausenstraBe 40-60, D-2300 Kiel. F.R.G. Received February 18, 1981 . Accepted March 2, 1981
Summary Sucrose solutions introduced into the vessels of isolated shoots of POPUlU5 show a dearcut decrease in sucrose and a concomitant increase in hexose coment. The total content of sucrose and hexose, however, decreases at a rate of 0.5 to 3.2 mg hexose/ mt tracheal sap' h. This indicates sucrose hydrolysis in the free space and the presence of a hexose uptake system in the secondary xylem. The appropriate flux rate between ray parenchyma cell s and vessel s is computed to be 230 to 11 85 pmol cm-2 min-I , and 460 to 2370 pmal cm- 2 min- t between contact cells and vessels.
Key w ords: Populus canadensis, xylem, tracheal sap, hexose, sucrose h ydrolysis.
Introd uction The uptake of sugars by cells and tissues has increasingly attracted the attention of transport physiologists during the past two decades. One of the best studied systems in eukaryotic p lant cells is the hexose-proton-cotransport system in Chiarella (e.g. Komar, 1973). In higher plants, results have been obtained mainly using tissue slices and leave disks (e.g. Bowen, 1972; Humphreys, 1973; Giaquinta, 1976; Stein and Willenbrink, 1976) or cells in suspension cultures (e.g. Ma retzki and Thorn, 1972) but rarely using whole organs like cotyledons (Komor, 1977) or stem internodes (Van Bel, 1976). When sucrose is being loaded into the phloem, i.e. into the sieve element-companion cell complex, it apparently is taken up from the free space without prior hydrolysis (Geiger et a1., 1973; Giaquinta, 1976, 1980; Fondy and Geiger, 1977). In contrast, sucrose entering the storage parenchyma in sugar cane from the vascu lar tissue is found to be hydrolysed by invertase in the free space before it is taken up as hexoses by a carrier mediated active transport system for glucose (e.g. Bowen and Hunter, 1972; Gayler and Glasziou, 1972). In the tracheal sap of many tree species a comparatively high content of sucrose Z. P/lanzenphysiol. Bd. 103. S. 165- 168. 1981.
166
JORG
J.
SAUTER
Or hexoses is encountered in late winter or early spring (d. Sauter, 1980). The present results on Populus give evidence for the reabsorption of these sugars from the vessels into the adjacent parenchyma cells and for the hydrolysis of the sucrose prior to its uptake.
Materials and Methods One- to 4-year-old shoots of an adult tree of Populus X canadensis Moench in the Botanical Garden of Kiel University had been collected in the winter stage (November until February). They have been cut into sections of 10 to 20 em in length which then were perfused with sucrose solutions (2 and 5 Ofo w / v in tap water), applying low pressure at their morphological upper end. After 2 to 24 h of storage at 21 and 2 °C (± 1°). the tracheal sap was collected from these shoot sections again and was analysed colorimetrically for its content of sucrose and hexoses using the Somogyi-method (d. Sauter, 1980). The amount of sugar that had been taken up during the experiment is calculated from the difference in concentration of the sucrose solution introduced into the vessels and the combined content of sucrose and hexoses recovered in the tracheal sap after the storage. Care had been taken to saturate the apoplast at the beginning with the perfused solution and also to avoid contamination with phloem constituents when the tracheal sap had been collected again.
Results and Discussion 1. The system under investigation The system used is very similar to that described previously for Salix (cf. Fig. 3 in Sauter 1980): In the wood of POpt~lu$ microporous vessels (diameter about SO .urn) are embedded in a ground tissue consisting of dead wood fibers. The only living cells that are bordering the vessels are the wood ray parenchyma cells, as axial wood parenchyma is almost completely absent. From measurements on radial longitudinal t issue sections it is deduced that about 25 % of the total outer surface of the vessels is adjacent to ray parenchyma cells of which about so % are specialized contact cells (cf. Sauter, 1972).
2. The uptake of sucrose At 21 °C: When sucrose solutions of 2 and 5 010 (w/v) are introduced into the
vessels of shoot sections, both, the sucrose content and the sucrose/hexose ratio are changing most drastically after a storage period of 2 to 24 h at room temperature. From table 1 it is seen: First, that the sucrose content decreases in the tracheal sap within 24 h from 2 0/0 ·t o 0.11 0/0. and from 5 010 to 0.17 0/0. The sucrose thus almost completely disappears during this period. Second, hexoses are appearing instead in considerable amounts, e.g. 0.64 and 2.33 0/0, respectively. They must be considered to be the result of sucrose hydrolysis by a free space invertase, as almost no hexose was present before in the perfused solution. Additional experiments in which tracheal sap samples were incubated in vitro with sucrose solutions of identical concentrations showed no invertase activity so far. We assume, therefore, that an invertase which obviously is not diffusing Z. Pfianzenphy,ioi. Bd. 103. S. 165- 168. 1981.
Sucrose uptake in the xylem
167
Table I: The sucrose content of solutions introduced into the vessels of shoot sections of Populus at the s~ and h~ = sucrose beginning (Sb = 2 and 5 % w / v) and after 2, 5, and 24 hours of storage (= t) at 21 and hexose content, respectively, in % w/ v at the end of the storage period. The uptake rate is given in mg hexose units per ml vessel water per hour.
0c.
,
(h)
Sb
S,
h,
s~ + h~
uptake rate (mgml - 'h - ' )
.(% w/ v)
24 24
2 5
0.11 0. 17
0.64 2.33
0.75 2.50
0.52 1.04
5 5
2 5
0.43 2.52
1.17 1.28
1.60 3.80
0.80 2.40
2
2
1.00
0.49
1.49
2.50
into the vessel water is located extraplasmatically in the cell wall between ray cells and vessels. Such a free space invertase is well known from studies in sugar cane (Samer et aI., 1963; Bowen and Hunter, 1972). Third, adding the sucrose and hexose content that are found at the end of the experiments reveals that a considerable amount of the introduced sucrose has disappeared. Because the free space has been sufficiently saturated with the perfused solution at the beginning of the experiment, the missing amount of sugars 1S believed to have been taken up into the symplastic compartment, i.e. the ray cells bordering the vessels. Forth, an uptake of 0.52 and 1.04 mg hexose-units/ ml vessel water' h can be calculated for the 2 % and the 5 % sucrose solutions, respectively (table 1). From the 5 hours experiment (see table 1) two additional res ults are of interest: Although the sucrose has been split again readily into hexoses, a considerable content of sucrose is still left in the tracheal sap, in cOntrast (0 the 24 h experiment. This indicates that the 5 h incubation period is not long enough for the invertase to hydrolyse the sucrose more completely, or, that there is a feed-back inhibition of the invertase by the hexoses that have been taken up only in part. Nevertheless, uptake clearly occured with an uptake rate that is even higher than in the 24 h experiment, e.g. 0.80 and 2.40 mg ml-' h-' (table 1). The 2 hours experiment finall y shows that even at this short intervall about half of the sucrose introduced into the vessels is hydrolysed and that a clear-cut amount of the resulting hexoses has already been taken up. On the other hand, this incubation time is thought to be less favourable because only half of the sucrose is split and also because any loss into the free space is affecting relatively more the calculated uptake rates for the sucrose. At 2°C: When the uptake rates at 21 ° and at 2 °C are compared in experiments running for 2, 4, and 24 h, it is seen that always less than 50 % of the uptake at room temperature is found at 2 °C (table 2). The low uptake rate thereby is definitively not caused by a shortage of hexoses as the invertase always proved to be still quite active at this low temperature. Z. P/lanzenphysiol. Bd. 103. S. 165-168. 1981.
168
JORG
J.
SAUTER
Tab le 2: The uptake rates in hexose units (mg ml - I h - I) in the secondary xylem of Populus shoots at 21 ° c and 2 0c. A sucrose so lution o f 2 % w/ v was introduced at the beginning of the 2,4, and 24 hours storage period s. ur ll11 = uptake rate at 2 °C in % of the uptake rate at 21 0c. 2h
4h
24h
21 °C 2°C
2.50 0.95
1.72
0.85
0.52 0.22
url21
38%
49'''10
42 'X,
3. The calculated flux rates In the shoot sections taken from November until February the following uptake rates were obtained (in mg hexose/ ml vessel water' h): 0 .5 after 24 h, 0.7 to 1.9 after 5 h, and 2.5 to 3.2 after 2 h incubation at 21 °C, when the concentration of the perfused sucrose solution was 2 % (w/ v). If the mg hexose is converted to mol and 800 em! outer surface is introduced for a vessel containing 1 ml solution (d. Sauter, 1980), a flux rate of 57.8 to 370.4 pmol cm-! min-I is obtained for the uptake rate of 0.5 to 3.2 mg ml- 1 h- ' . The minimal flux rate of hexose between ray cells and vessels then becomes 230 to 1185 pmol cm-! min- t , because the permeation area between both is only about 25 % of the outer vessel surface (see above). If the uptake of sucrose after its hydrolysis to hexoses is not achieved by all of the ray cells but only by the specialized contact cells (Sauter, 1972, 1980; Sauter et al., 1973) these flux rates again increase at least by a factor of two. The height of these flux rates are in good accordance with the rates obtained in other systems (e.g. Komor, 1977, and literature cited therein) and thus can be taken to indicate the presence of a 'hexose uptake system in the secondary xylem of Populus. References
J. J.
L Plant Physio!. 49, 82 (1972). E. and J. E. HUNTER: Plant Physio!. 49, 789 (1972). FONDY, B. R. and D. R. GEIGER: Plant Physiol. 59, 953 (1977). GAYLER, K. R . and K. T. GLASZIOU: Plant Physiol. 49, 563 (1972). GEIGER, D. R., R. T. GIAQUINTA, S. A. SOVoONICK, and R. J. FELLOWS : Plant Physio!. 52, 585 (1973). GIAQUINTA, R.: Plant Physio!. 57, 872 (1976). - Ber. D eutsch. Bot. Ges. 93, 187 (1980). HUMPHREYS, T. E.: Phytochemist ry 12,1211 (1973). KOM OR, E.: FEBS-Lettcrs 38,16 (1973) . - Planta 137, 119 (1977). MARETZKJ, A. and M. THOM: Plant Physiol. 49,177 (1972). SACHER, J. A., M. D. HATCH, and K . T. GLASZIOU: Plant Physio!. 38, 348 (1963). SAUTER, J. J.: Z. Pflanzenphysio!. 67,135 (1972). - Z. Pflanzenphysio!. 98, 377 (1980). SAUTER, J. J., W. hEN, and M. H. ZIMMERMANN: Can. J. Bot. 51, 1 (1973). STEIN, M. and J. WILLENBRINK: Z. Pflanzenphysiol. 79, 310 (1976). VAN BEL, A. J. E . : Plant Physio!. 57, 911 (1976). BOWEN, BOWEN,
Z. P/lanzenphysiol. Bd. 103. S. 165-168. 1981.
Sucrose Uptake i n the Xylem of Populus JORG J. SAUTER
Botan ismes Institut del' Universitat Kiel, OlshausenstraBe 40-60, D-2300 Kiel. F.R.G. Received February 18, 1981 . Accepted March 2, 1981
Summary Sucrose solutions introduced into the vessels of isolated shoots of POPUlU5 show a dearcut decrease in sucrose and a concomitant increase in hexose coment. The total content of sucrose and hexose, however, decreases at a rate of 0.5 to 3.2 mg hexose/ mt tracheal sap' h. This indicates sucrose hydrolysis in the free space and the presence of a hexose uptake system in the secondary xylem. The appropriate flux rate between ray parenchyma cell s and vessel s is computed to be 230 to 11 85 pmol cm-2 min-I , and 460 to 2370 pmal cm- 2 min- t between contact cells and vessels.
Key w ords: Populus canadensis, xylem, tracheal sap, hexose, sucrose h ydrolysis.
Introd uction The uptake of sugars by cells and tissues has increasingly attracted the attention of transport physiologists during the past two decades. One of the best studied systems in eukaryotic p lant cells is the hexose-proton-cotransport system in Chiarella (e.g. Komar, 1973). In higher plants, results have been obtained mainly using tissue slices and leave disks (e.g. Bowen, 1972; Humphreys, 1973; Giaquinta, 1976; Stein and Willenbrink, 1976) or cells in suspension cultures (e.g. Ma retzki and Thorn, 1972) but rarely using whole organs like cotyledons (Komor, 1977) or stem internodes (Van Bel, 1976). When sucrose is being loaded into the phloem, i.e. into the sieve element-companion cell complex, it apparently is taken up from the free space without prior hydrolysis (Geiger et a1., 1973; Giaquinta, 1976, 1980; Fondy and Geiger, 1977). In contrast, sucrose entering the storage parenchyma in sugar cane from the vascu lar tissue is found to be hydrolysed by invertase in the free space before it is taken up as hexoses by a carrier mediated active transport system for glucose (e.g. Bowen and Hunter, 1972; Gayler and Glasziou, 1972). In the tracheal sap of many tree species a comparatively high content of sucrose Z. P/lanzenphysiol. Bd. 103. S. 165- 168. 1981.
166
JORG
J.
SAUTER
Or hexoses is encountered in late winter or early spring (d. Sauter, 1980). The present results on Populus give evidence for the reabsorption of these sugars from the vessels into the adjacent parenchyma cells and for the hydrolysis of the sucrose prior to its uptake.
Materials and Methods One- to 4-year-old shoots of an adult tree of Populus X canadensis Moench in the Botanical Garden of Kiel University had been collected in the winter stage (November until February). They have been cut into sections of 10 to 20 em in length which then were perfused with sucrose solutions (2 and 5 Ofo w / v in tap water), applying low pressure at their morphological upper end. After 2 to 24 h of storage at 21 and 2 °C (± 1°). the tracheal sap was collected from these shoot sections again and was analysed colorimetrically for its content of sucrose and hexoses using the Somogyi-method (d. Sauter, 1980). The amount of sugar that had been taken up during the experiment is calculated from the difference in concentration of the sucrose solution introduced into the vessels and the combined content of sucrose and hexoses recovered in the tracheal sap after the storage. Care had been taken to saturate the apoplast at the beginning with the perfused solution and also to avoid contamination with phloem constituents when the tracheal sap had been collected again.
Results and Discussion 1. The system under investigation The system used is very similar to that described previously for Salix (cf. Fig. 3 in Sauter 1980): In the wood of POpt~lu$ microporous vessels (diameter about SO .urn) are embedded in a ground tissue consisting of dead wood fibers. The only living cells that are bordering the vessels are the wood ray parenchyma cells, as axial wood parenchyma is almost completely absent. From measurements on radial longitudinal t issue sections it is deduced that about 25 % of the total outer surface of the vessels is adjacent to ray parenchyma cells of which about so % are specialized contact cells (cf. Sauter, 1972).
2. The uptake of sucrose At 21 °C: When sucrose solutions of 2 and 5 010 (w/v) are introduced into the
vessels of shoot sections, both, the sucrose content and the sucrose/hexose ratio are changing most drastically after a storage period of 2 to 24 h at room temperature. From table 1 it is seen: First, that the sucrose content decreases in the tracheal sap within 24 h from 2 0/0 ·t o 0.11 0/0. and from 5 010 to 0.17 0/0. The sucrose thus almost completely disappears during this period. Second, hexoses are appearing instead in considerable amounts, e.g. 0.64 and 2.33 0/0, respectively. They must be considered to be the result of sucrose hydrolysis by a free space invertase, as almost no hexose was present before in the perfused solution. Additional experiments in which tracheal sap samples were incubated in vitro with sucrose solutions of identical concentrations showed no invertase activity so far. We assume, therefore, that an invertase which obviously is not diffusing Z. Pfianzenphy,ioi. Bd. 103. S. 165- 168. 1981.
Sucrose uptake in the xylem
167
Table I: The sucrose content of solutions introduced into the vessels of shoot sections of Populus at the s~ and h~ = sucrose beginning (Sb = 2 and 5 % w / v) and after 2, 5, and 24 hours of storage (= t) at 21 and hexose content, respectively, in % w/ v at the end of the storage period. The uptake rate is given in mg hexose units per ml vessel water per hour.
0c.
,
(h)
Sb
S,
h,
s~ + h~
uptake rate (mgml - 'h - ' )
.(% w/ v)
24 24
2 5
0.11 0. 17
0.64 2.33
0.75 2.50
0.52 1.04
5 5
2 5
0.43 2.52
1.17 1.28
1.60 3.80
0.80 2.40
2
2
1.00
0.49
1.49
2.50
into the vessel water is located extraplasmatically in the cell wall between ray cells and vessels. Such a free space invertase is well known from studies in sugar cane (Samer et aI., 1963; Bowen and Hunter, 1972). Third, adding the sucrose and hexose content that are found at the end of the experiments reveals that a considerable amount of the introduced sucrose has disappeared. Because the free space has been sufficiently saturated with the perfused solution at the beginning of the experiment, the missing amount of sugars 1S believed to have been taken up into the symplastic compartment, i.e. the ray cells bordering the vessels. Forth, an uptake of 0.52 and 1.04 mg hexose-units/ ml vessel water' h can be calculated for the 2 % and the 5 % sucrose solutions, respectively (table 1). From the 5 hours experiment (see table 1) two additional res ults are of interest: Although the sucrose has been split again readily into hexoses, a considerable content of sucrose is still left in the tracheal sap, in cOntrast (0 the 24 h experiment. This indicates that the 5 h incubation period is not long enough for the invertase to hydrolyse the sucrose more completely, or, that there is a feed-back inhibition of the invertase by the hexoses that have been taken up only in part. Nevertheless, uptake clearly occured with an uptake rate that is even higher than in the 24 h experiment, e.g. 0.80 and 2.40 mg ml-' h-' (table 1). The 2 hours experiment finall y shows that even at this short intervall about half of the sucrose introduced into the vessels is hydrolysed and that a clear-cut amount of the resulting hexoses has already been taken up. On the other hand, this incubation time is thought to be less favourable because only half of the sucrose is split and also because any loss into the free space is affecting relatively more the calculated uptake rates for the sucrose. At 2°C: When the uptake rates at 21 ° and at 2 °C are compared in experiments running for 2, 4, and 24 h, it is seen that always less than 50 % of the uptake at room temperature is found at 2 °C (table 2). The low uptake rate thereby is definitively not caused by a shortage of hexoses as the invertase always proved to be still quite active at this low temperature. Z. P/lanzenphysiol. Bd. 103. S. 165-168. 1981.
168
JORG
J.
SAUTER
Tab le 2: The uptake rates in hexose units (mg ml - I h - I) in the secondary xylem of Populus shoots at 21 ° c and 2 0c. A sucrose so lution o f 2 % w/ v was introduced at the beginning of the 2,4, and 24 hours storage period s. ur ll11 = uptake rate at 2 °C in % of the uptake rate at 21 0c. 2h
4h
24h
21 °C 2°C
2.50 0.95
1.72
0.85
0.52 0.22
url21
38%
49'''10
42 'X,
3. The calculated flux rates In the shoot sections taken from November until February the following uptake rates were obtained (in mg hexose/ ml vessel water' h): 0 .5 after 24 h, 0.7 to 1.9 after 5 h, and 2.5 to 3.2 after 2 h incubation at 21 °C, when the concentration of the perfused sucrose solution was 2 % (w/ v). If the mg hexose is converted to mol and 800 em! outer surface is introduced for a vessel containing 1 ml solution (d. Sauter, 1980), a flux rate of 57.8 to 370.4 pmol cm-! min-I is obtained for the uptake rate of 0.5 to 3.2 mg ml- 1 h- ' . The minimal flux rate of hexose between ray cells and vessels then becomes 230 to 1185 pmol cm-! min- t , because the permeation area between both is only about 25 % of the outer vessel surface (see above). If the uptake of sucrose after its hydrolysis to hexoses is not achieved by all of the ray cells but only by the specialized contact cells (Sauter, 1972, 1980; Sauter et al., 1973) these flux rates again increase at least by a factor of two. The height of these flux rates are in good accordance with the rates obtained in other systems (e.g. Komor, 1977, and literature cited therein) and thus can be taken to indicate the presence of a 'hexose uptake system in the secondary xylem of Populus. References
J. J.
L Plant Physio!. 49, 82 (1972). E. and J. E. HUNTER: Plant Physio!. 49, 789 (1972). FONDY, B. R. and D. R. GEIGER: Plant Physiol. 59, 953 (1977). GAYLER, K. R . and K. T. GLASZIOU: Plant Physiol. 49, 563 (1972). GEIGER, D. R., R. T. GIAQUINTA, S. A. SOVoONICK, and R. J. FELLOWS : Plant Physio!. 52, 585 (1973). GIAQUINTA, R.: Plant Physio!. 57, 872 (1976). - Ber. D eutsch. Bot. Ges. 93, 187 (1980). HUMPHREYS, T. E.: Phytochemist ry 12,1211 (1973). KOM OR, E.: FEBS-Lettcrs 38,16 (1973) . - Planta 137, 119 (1977). MARETZKJ, A. and M. THOM: Plant Physiol. 49,177 (1972). SACHER, J. A., M. D. HATCH, and K . T. GLASZIOU: Plant Physio!. 38, 348 (1963). SAUTER, J. J.: Z. Pflanzenphysio!. 67,135 (1972). - Z. Pflanzenphysio!. 98, 377 (1980). SAUTER, J. J., W. hEN, and M. H. ZIMMERMANN: Can. J. Bot. 51, 1 (1973). STEIN, M. and J. WILLENBRINK: Z. Pflanzenphysiol. 79, 310 (1976). VAN BEL, A. J. E . : Plant Physio!. 57, 911 (1976). BOWEN, BOWEN,
Z. P/lanzenphysiol. Bd. 103. S. 165-168. 1981.