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Evidence for Sucrose Efflux and Hexose Uptake in the Xylem of Salix
Short Communication Evidence for Sucrose Efflux and Hexose Uptake in the Xylem of Salix JORG
J. SAUTER
Botaniscnes Institut de r Universidit Kiel, Olshausenstra6e 40-60, D-2300 Kiel, F.R.G. Received March 22,1981 . Accepted March 27,198 1
Summary Shoot sections of Salix taken in mid-winter stage show renewed sucrose accumulation in the tracheal sap when the tracheal sap is exchanged against tap water. Influx from the symplast proceeds at a rate of about 65 pmcl cm-! min- t. When the t racheal sap is exchanged against sucrose solutions (1.2 and 5 °/0 w / v), reabsorption of sucrose into the symplast is observed at 21 °, but not at 2 °C. As the decrease in sucrose content is always paralleled initially by a considerable increase and then by a continuous decrease in hexose content, uptake of sucrose is suggested to proceed via a hexose-uptake system. Hexoses are found to originate in the tracheal sap on the account of the sucrose at 2° and at 21 °e, while their uptake is only observed at 21 °C.
Key words: Salix, xylem, tracheal sap, hexose, sucrose.
Introduction In late winter, the tracheal sap of willow is found to be unusually rich in sucrose (Sauter, 1980). Shortly before the blossoming of the male catkins, the sucrose content reaches as much as 3 to 5 % (w/v). Amids and more than 20 different amino acids also appear in the tracheal sap in considerable amounts exactly during this period (Sauter, 1981 a). Both resu lts emphasize the importance of the xylem pathway for the translocation of organic materials in early spring in willow. Little, however, is known about the process of reentrance of these substances into the symplast (e.g. Van Bel, 1974 a, h, 1976). The sucrose is known to he either hydrolysed by an invertase in the free space before it is taken up as hexose, e.g. into the storage parenchyma of sugar cane (d. Bowen and Hunter, 1972; Gayler and Glasziou, 1972), or it may be taken up unchanged, e.g. when it is loaded into ~th e phloem. (cf. Giaquinta, 1976, 1980) . Z. Pflanzenphysiol. Bd. 103. S. 183-187. 1981.
184
JORG
J.
SAUTER
In willow, the sucrose content decreases sharply in the vessels of catkin-bearing shoots during blossoming, indicating an uptake into the symplast. Because the hexose content thereby was found to increase slightly but significantly (Sauter, 1980) it is suspected that the sucrose is taken up via a hexose-uptake system after its hydrolysis. Further evidence for the presence of a hexose-uptake system in the secondary xylem of Salix is obtained from the preliminary experiments described in this paper.
Material and Methods Two- to three-year-old shoots of a male Salix X smithiana Willd. had been collected in mid-winter stage from an adult tree in the Botanical Garden of Kiel University. From these shoots, sections of 15 to 20 cm in length had been prepared which then were perfused with tap water or sucrose solutions of different concentrations (see results). The tracheal sap was collected from these shoot sections again afte r 1, 24, and 96 h of storage at 21 ° or 2 °C (± 1°) and analysed for its content of sucrose and hexoses as described previously (Sauter, 1980, 1981 b). The appropriate solution had been newly infused at the begin of each storage period. Results and Discussion In mid-January, when the shoots were collected, already 1.2 % (w/ v) of sucrose had been accumulated in the tracheal sap under outdoor conditions. The hexose content, in contrast, is very low and lies around 0.04 0/0. In the fo llowing experiments the tracheal sap of t hese shoots was exchanged either against tap water for studying the influx of sucrose or agai nst a sucrose solution of 1.2 % (= the concentration that is found in nature at this stage) or 5 % ( = th e maximum concentration that is found at the begin of blossoming) for studying the possible reabsorption of sucrose from the vessels. At the end of the exchange procedure and after 1, 24, and 96 h of storage at 21 ° or 2 °C (± 1°), the tracheal sap was collected and ana lysed again for its content of sucrose and hexoses. T he results are summarized in table 1. Table 1: The content of sucrose (a) and hexoses (b) in Ufo (w/v) in the tracheal sap of shoot sections of Salix after infusion of tap water or sucrose solutions (1.2 and 5.0% w/v) and subsequent storage forO, 1,24, and 96h at 21°e and 2°C, respectively. storage at 21°e
solution perfused
storage at 2°e
0
1
24
96 (h)
0
1
24
96 (h)
tap water
a b
0.11 0.00
0.24
0.64 0.08
0048 0.03
0.15 0.02
0.13 0.03
0.13 0.06
0.39 0.16
sucrose 1.2 %
a b
1. 14 0.02
0.60 0.28
0.88 0.22
0.89 0.07
1.1 5 0.03
1.05 0.04
1.08 0.17
0048 0.50
sucrose 5.0%
a b
4.28 0.02
3.62 0.14
2.98 0.78
2.54 0.08
om
4.60
4.44 0.04
4.08 0.54
2.20 2.39
Z. Pjianzenphysioi. Bd. 103. S. 183-187. 1981.
Sucrose efflux and hexose uptake
185
a) Infusion of tap water
When tap water is introduced into the vessels at this stage, sucrose accumu lates therein again up to 0.64 % within 24 h at 21 °C. This corresponds to an influx of 65 pmol sucrose cm- 2 min- t when the permeation area is taken as the area between parenchyma cells and vessels which is t/4 of the outer vessel surface (d. Sauter, 1980). Very similar flux rates had been obtained in a previous study (Sauter, 1980). After additional storage for 4 days a't 21 °C, no further increase occurs. In contrast, sucrose content is lower again, e.g. 0,48 %. This indicates either reabsorption of sucrose from the vessels or shortage of sucrose in the symplast that could permeate into the apoplast. Because 4 days of storage at 21 °C are found to induce a most conspicuous resynthesis of starch in the parenchymatous tissues of these shoot sections (Sauter, unpublished results; d. Sauter, 1967) which proceeds at least in part on the account of the intracellular sucrose pool, both of the given explanations are feasible. In contrast to the sucrose, the content of hexoses remains comparatively low under these conditions, indicating that sugar efflux is as sucrose and not as hexoses. From the shoot sections kept at 2 °C two results are of interest: First, much less sucrose is accumulating in the tracheal sap during the first 24 h, e.g. only 0.13 0/0 as compared to 0.68 % at 21 °C. Second, sucrose accumulation clearly continues at this low temperature during the 4 days of storage, in contrast to the experiment running at 21 DC. This increasing sucrose accumu laotion in the vessels is only possible if no reabsorption into the symplast on the one hand, and no shortage in sucrose to be released on the other hand is impeding this process. An additional indication for the missing reabsorption under these conditions is the comparatively higher hexose content a't 2 °C as compared to 21 °C (see also below). b) Infusion of sucrose solution (1.2 % w/v)
When a sucrose solution of the same concentra.tion that is found under outdoor conditions is introduced into the vessels, remarkable differences are obtained after the storage at 2° and 21 °C. At 21 DC, the sucrose content of the tracheal sap decreases rapidly from 1.2 to about 0.88°/0. Concomitantly a sharp rise in hexose content is observed, reaching 0.22010 after 24 h, but decreasing again to 0.07°10 after 4 days. While the decrease in sucrose content can be explained by the hydrolytical splitting of sucrose to hexoses, ·the decrease in hexose content after 4 days must be attributed to the uptake of hexoses into the symplastic compartment. This view is further supported by the results obtained at 2 °C and in particular by the results obtained with the 5 % sucrose solution. At 2 DC, the sucrose content decreases only from 1.2 to 1.08 % after 24 h, whi le 0.17 % of hexoses are arising at the same time. The decrease in sucrose content thus is clearly slower than at 21 °C. After 4 days, however, only 0,48 % of sucrose are Z. Pjlanzenphysiol. Bd. 103. S. 183-187. 1981.
186
JORG
J.
SAUTER
left while the content of hexoses now reaches as much as 0.50 0 / 0. This result is of particular interest, as it proves, first, that hydrolysis of sucrose also proceeds at this low temperature within the shoots, and second, that the originating hexoses obviously cannot be taken up into the symplast, and therefore are accumulating relatively in the tracheal sap, in contrast to the experiment at 21 °e. c) Infusion of sucrose solution (5.0 % w/v)
Finally a sucrose solution of 5 010 (w/v) was introduced into the shoot sections as such a high sucrose concentration also was found in nature, though only at the stage before blossoming in late February and early March (cf. Sauter, 1980). At 21 °e, the sucrose content decreases rapidly in the tracheal sap, e.g. from 5 % to 2.98 and 2.52 % after 24 hand 4 days, respectively. Running parallel there is a very prominent increase in hexose content, reaching as much as 0.78 0/ 0 after 24 h. It undoubtedly is caused by the comparatively rapid splitting of the sucrose that is supplied to the vessels and the missing of a similar rapid uptake of the resulting hexoses into the symplast within the first 24 h. A similar extracellular hydrolysis of sucrose is known from its uptake in sugar cane storage parenchyma (d. Bowen and Hunter, 1972). After 4 days, however, uptake of hexoses becomes much more evident, as only 0.08 010 of hexoses are left in the tracheal sap in addi,t ion to 2.52 °/0 of sucrose. Similarly to the former experiment with the 1.2 0 10 sucrose solution, it again appears that the hexoses resulting from sucrose hydrolysis are more completely taken up only after 4 days. Whether the uptake system for hexoses is not operating efficiently during the first 24 h when the stems are collected in deep winter stage or whether the uptake is impaired by the height of the intracellular sucrose pool at this stage, remains to be disclosed in further experiments. At 2 °e, two striking results are obtained. First, sucrose hydrolysis again is found to proceed clearly at this low temperature, delivering 0,54 and 2,39 % of hexoses in the tracheal sap after 24 hand 4 days, respectively, Second, because as much as 2.39°10 of hexoses are accumulating after 4 days, it mUSt be concluded that there is almost no hexose uptake a.t this temperature, in contrast to the experiment at 21 °C in which only 0.08 010 of hexoses were left. The results obtained at 2 °e thus are good prove, so far, that the view of a hydrolytical splitting of the sucrose within the vessels and subsequent uptake of the hexoses into the symplastic compartment, e.g, into the ray parenchyma bordering the vessels, is correct, The flux rates and the characteristics of this system in the secondary xylem will be studied in further experiments.
References
J. E. and J. E, HUNTER: Sugar transport in immature internodal tissue of sugar cane. II. Mechanism of sucrose transport. Plant Physio!. 49, 789- 793 (1972).
BOWEN,
Z. PJlanzenphysiol. Bd. 103. S. 183- 187. 1981.
Sucrose efflux and hexose uptake
187
GAYLER, K . R. and K. T. GLASZIOU: Sugar accumulation in sugar cane. Carrier-mediated active transport of glucose. Plant Physiol. 49, 563-568 (1972). GIAQUINTA, R.: Evidence for phloem loading from the apoplast. Chemical modification of membrane sulfhydryl groups. Plant Physiol. 57, 872-875 (1976) . - Mechanism and control of phloem loading of sucrose. Ber. Deutsch. Bot. Ges. 93, 187- 201 (1980) . SAUTER, J. J.: Der EinfluB verschiedener Temperaturen auf die Reservestarke in parenchymatischen Geweben von BaumsproBachsen. Z. Pflanzenphysiol. 56, 340-352 (1967). - Seasonal variation of sucrose content in the xylem sap of Salix. Z. Pflanzenphysiol. 98,377-391 (1980). - Seasonal variation of amino acids and ami des in the xy lem sap of Salix. Z. Pflanzenphysiol.l0l, 309-411 (1981 a). - Sucrose uptake in the xylem of Populus. Z. Pflanzenphysiol. 103, 165-168 (1981) . VAN BEL, A. J. E.: The absorption of L-a-alanine and L-a-aminoisobutyric acid during their movement through the xylem vessels of tomato stem segments. Acta Bot. Neerl. 23, 305-313 (1974 a). - Different rates translocation of 14C-L-a-alanine (U) and tritiated water in the xylem vessels of tomato plants. Acta Bot. Neerl. 23,, 715-722 (1974 b). - Different mass transfer rates of labeled sugars and tritiated water in xylem vessels and their dependency on metabolism. Plant Physiol. 57, 911-914 (1976).
Z. Pflanzenphysiol. Bd. 103. S. 183-187. 1981.
J. SAUTER
Botaniscnes Institut de r Universidit Kiel, Olshausenstra6e 40-60, D-2300 Kiel, F.R.G. Received March 22,1981 . Accepted March 27,198 1
Summary Shoot sections of Salix taken in mid-winter stage show renewed sucrose accumulation in the tracheal sap when the tracheal sap is exchanged against tap water. Influx from the symplast proceeds at a rate of about 65 pmcl cm-! min- t. When the t racheal sap is exchanged against sucrose solutions (1.2 and 5 °/0 w / v), reabsorption of sucrose into the symplast is observed at 21 °, but not at 2 °C. As the decrease in sucrose content is always paralleled initially by a considerable increase and then by a continuous decrease in hexose content, uptake of sucrose is suggested to proceed via a hexose-uptake system. Hexoses are found to originate in the tracheal sap on the account of the sucrose at 2° and at 21 °e, while their uptake is only observed at 21 °C.
Key words: Salix, xylem, tracheal sap, hexose, sucrose.
Introduction In late winter, the tracheal sap of willow is found to be unusually rich in sucrose (Sauter, 1980). Shortly before the blossoming of the male catkins, the sucrose content reaches as much as 3 to 5 % (w/v). Amids and more than 20 different amino acids also appear in the tracheal sap in considerable amounts exactly during this period (Sauter, 1981 a). Both resu lts emphasize the importance of the xylem pathway for the translocation of organic materials in early spring in willow. Little, however, is known about the process of reentrance of these substances into the symplast (e.g. Van Bel, 1974 a, h, 1976). The sucrose is known to he either hydrolysed by an invertase in the free space before it is taken up as hexose, e.g. into the storage parenchyma of sugar cane (d. Bowen and Hunter, 1972; Gayler and Glasziou, 1972), or it may be taken up unchanged, e.g. when it is loaded into ~th e phloem. (cf. Giaquinta, 1976, 1980) . Z. Pflanzenphysiol. Bd. 103. S. 183-187. 1981.
184
JORG
J.
SAUTER
In willow, the sucrose content decreases sharply in the vessels of catkin-bearing shoots during blossoming, indicating an uptake into the symplast. Because the hexose content thereby was found to increase slightly but significantly (Sauter, 1980) it is suspected that the sucrose is taken up via a hexose-uptake system after its hydrolysis. Further evidence for the presence of a hexose-uptake system in the secondary xylem of Salix is obtained from the preliminary experiments described in this paper.
Material and Methods Two- to three-year-old shoots of a male Salix X smithiana Willd. had been collected in mid-winter stage from an adult tree in the Botanical Garden of Kiel University. From these shoots, sections of 15 to 20 cm in length had been prepared which then were perfused with tap water or sucrose solutions of different concentrations (see results). The tracheal sap was collected from these shoot sections again afte r 1, 24, and 96 h of storage at 21 ° or 2 °C (± 1°) and analysed for its content of sucrose and hexoses as described previously (Sauter, 1980, 1981 b). The appropriate solution had been newly infused at the begin of each storage period. Results and Discussion In mid-January, when the shoots were collected, already 1.2 % (w/ v) of sucrose had been accumulated in the tracheal sap under outdoor conditions. The hexose content, in contrast, is very low and lies around 0.04 0/0. In the fo llowing experiments the tracheal sap of t hese shoots was exchanged either against tap water for studying the influx of sucrose or agai nst a sucrose solution of 1.2 % (= the concentration that is found in nature at this stage) or 5 % ( = th e maximum concentration that is found at the begin of blossoming) for studying the possible reabsorption of sucrose from the vessels. At the end of the exchange procedure and after 1, 24, and 96 h of storage at 21 ° or 2 °C (± 1°), the tracheal sap was collected and ana lysed again for its content of sucrose and hexoses. T he results are summarized in table 1. Table 1: The content of sucrose (a) and hexoses (b) in Ufo (w/v) in the tracheal sap of shoot sections of Salix after infusion of tap water or sucrose solutions (1.2 and 5.0% w/v) and subsequent storage forO, 1,24, and 96h at 21°e and 2°C, respectively. storage at 21°e
solution perfused
storage at 2°e
0
1
24
96 (h)
0
1
24
96 (h)
tap water
a b
0.11 0.00
0.24
0.64 0.08
0048 0.03
0.15 0.02
0.13 0.03
0.13 0.06
0.39 0.16
sucrose 1.2 %
a b
1. 14 0.02
0.60 0.28
0.88 0.22
0.89 0.07
1.1 5 0.03
1.05 0.04
1.08 0.17
0048 0.50
sucrose 5.0%
a b
4.28 0.02
3.62 0.14
2.98 0.78
2.54 0.08
om
4.60
4.44 0.04
4.08 0.54
2.20 2.39
Z. Pjianzenphysioi. Bd. 103. S. 183-187. 1981.
Sucrose efflux and hexose uptake
185
a) Infusion of tap water
When tap water is introduced into the vessels at this stage, sucrose accumu lates therein again up to 0.64 % within 24 h at 21 °C. This corresponds to an influx of 65 pmol sucrose cm- 2 min- t when the permeation area is taken as the area between parenchyma cells and vessels which is t/4 of the outer vessel surface (d. Sauter, 1980). Very similar flux rates had been obtained in a previous study (Sauter, 1980). After additional storage for 4 days a't 21 °C, no further increase occurs. In contrast, sucrose content is lower again, e.g. 0,48 %. This indicates either reabsorption of sucrose from the vessels or shortage of sucrose in the symplast that could permeate into the apoplast. Because 4 days of storage at 21 °C are found to induce a most conspicuous resynthesis of starch in the parenchymatous tissues of these shoot sections (Sauter, unpublished results; d. Sauter, 1967) which proceeds at least in part on the account of the intracellular sucrose pool, both of the given explanations are feasible. In contrast to the sucrose, the content of hexoses remains comparatively low under these conditions, indicating that sugar efflux is as sucrose and not as hexoses. From the shoot sections kept at 2 °C two results are of interest: First, much less sucrose is accumulating in the tracheal sap during the first 24 h, e.g. only 0.13 0/0 as compared to 0.68 % at 21 °C. Second, sucrose accumulation clearly continues at this low temperature during the 4 days of storage, in contrast to the experiment running at 21 DC. This increasing sucrose accumu laotion in the vessels is only possible if no reabsorption into the symplast on the one hand, and no shortage in sucrose to be released on the other hand is impeding this process. An additional indication for the missing reabsorption under these conditions is the comparatively higher hexose content a't 2 °C as compared to 21 °C (see also below). b) Infusion of sucrose solution (1.2 % w/v)
When a sucrose solution of the same concentra.tion that is found under outdoor conditions is introduced into the vessels, remarkable differences are obtained after the storage at 2° and 21 °C. At 21 DC, the sucrose content of the tracheal sap decreases rapidly from 1.2 to about 0.88°/0. Concomitantly a sharp rise in hexose content is observed, reaching 0.22010 after 24 h, but decreasing again to 0.07°10 after 4 days. While the decrease in sucrose content can be explained by the hydrolytical splitting of sucrose to hexoses, ·the decrease in hexose content after 4 days must be attributed to the uptake of hexoses into the symplastic compartment. This view is further supported by the results obtained at 2 °C and in particular by the results obtained with the 5 % sucrose solution. At 2 DC, the sucrose content decreases only from 1.2 to 1.08 % after 24 h, whi le 0.17 % of hexoses are arising at the same time. The decrease in sucrose content thus is clearly slower than at 21 °C. After 4 days, however, only 0,48 % of sucrose are Z. Pjlanzenphysiol. Bd. 103. S. 183-187. 1981.
186
JORG
J.
SAUTER
left while the content of hexoses now reaches as much as 0.50 0 / 0. This result is of particular interest, as it proves, first, that hydrolysis of sucrose also proceeds at this low temperature within the shoots, and second, that the originating hexoses obviously cannot be taken up into the symplast, and therefore are accumulating relatively in the tracheal sap, in contrast to the experiment at 21 °e. c) Infusion of sucrose solution (5.0 % w/v)
Finally a sucrose solution of 5 010 (w/v) was introduced into the shoot sections as such a high sucrose concentration also was found in nature, though only at the stage before blossoming in late February and early March (cf. Sauter, 1980). At 21 °e, the sucrose content decreases rapidly in the tracheal sap, e.g. from 5 % to 2.98 and 2.52 % after 24 hand 4 days, respectively. Running parallel there is a very prominent increase in hexose content, reaching as much as 0.78 0/ 0 after 24 h. It undoubtedly is caused by the comparatively rapid splitting of the sucrose that is supplied to the vessels and the missing of a similar rapid uptake of the resulting hexoses into the symplast within the first 24 h. A similar extracellular hydrolysis of sucrose is known from its uptake in sugar cane storage parenchyma (d. Bowen and Hunter, 1972). After 4 days, however, uptake of hexoses becomes much more evident, as only 0.08 010 of hexoses are left in the tracheal sap in addi,t ion to 2.52 °/0 of sucrose. Similarly to the former experiment with the 1.2 0 10 sucrose solution, it again appears that the hexoses resulting from sucrose hydrolysis are more completely taken up only after 4 days. Whether the uptake system for hexoses is not operating efficiently during the first 24 h when the stems are collected in deep winter stage or whether the uptake is impaired by the height of the intracellular sucrose pool at this stage, remains to be disclosed in further experiments. At 2 °e, two striking results are obtained. First, sucrose hydrolysis again is found to proceed clearly at this low temperature, delivering 0,54 and 2,39 % of hexoses in the tracheal sap after 24 hand 4 days, respectively, Second, because as much as 2.39°10 of hexoses are accumulating after 4 days, it mUSt be concluded that there is almost no hexose uptake a.t this temperature, in contrast to the experiment at 21 °C in which only 0.08 010 of hexoses were left. The results obtained at 2 °e thus are good prove, so far, that the view of a hydrolytical splitting of the sucrose within the vessels and subsequent uptake of the hexoses into the symplastic compartment, e.g, into the ray parenchyma bordering the vessels, is correct, The flux rates and the characteristics of this system in the secondary xylem will be studied in further experiments.
References
J. E. and J. E, HUNTER: Sugar transport in immature internodal tissue of sugar cane. II. Mechanism of sucrose transport. Plant Physio!. 49, 789- 793 (1972).
BOWEN,
Z. PJlanzenphysiol. Bd. 103. S. 183- 187. 1981.
Sucrose efflux and hexose uptake
187
GAYLER, K . R. and K. T. GLASZIOU: Sugar accumulation in sugar cane. Carrier-mediated active transport of glucose. Plant Physiol. 49, 563-568 (1972). GIAQUINTA, R.: Evidence for phloem loading from the apoplast. Chemical modification of membrane sulfhydryl groups. Plant Physiol. 57, 872-875 (1976) . - Mechanism and control of phloem loading of sucrose. Ber. Deutsch. Bot. Ges. 93, 187- 201 (1980) . SAUTER, J. J.: Der EinfluB verschiedener Temperaturen auf die Reservestarke in parenchymatischen Geweben von BaumsproBachsen. Z. Pflanzenphysiol. 56, 340-352 (1967). - Seasonal variation of sucrose content in the xylem sap of Salix. Z. Pflanzenphysiol. 98,377-391 (1980). - Seasonal variation of amino acids and ami des in the xy lem sap of Salix. Z. Pflanzenphysiol.l0l, 309-411 (1981 a). - Sucrose uptake in the xylem of Populus. Z. Pflanzenphysiol. 103, 165-168 (1981) . VAN BEL, A. J. E.: The absorption of L-a-alanine and L-a-aminoisobutyric acid during their movement through the xylem vessels of tomato stem segments. Acta Bot. Neerl. 23, 305-313 (1974 a). - Different rates translocation of 14C-L-a-alanine (U) and tritiated water in the xylem vessels of tomato plants. Acta Bot. Neerl. 23,, 715-722 (1974 b). - Different mass transfer rates of labeled sugars and tritiated water in xylem vessels and their dependency on metabolism. Plant Physiol. 57, 911-914 (1976).
Z. Pflanzenphysiol. Bd. 103. S. 183-187. 1981.