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Poplar Storage Protein in Xylem Sap
J.PlantPhysiol. Vol. 137.pp. 763-764(1991)
Short Conlnlunication
Poplar Storage Protein in Xylem Sap BARBARA VAN CLEVE, JOACHIM JUST,
and JORG J.
SAUTER
Botanisches Institut der Universitat Kiel, OlshausenstraBe 40, D-2300 Kiel, Federal Republic of Germany Received November 7,1990 . Accepted November 27,1990
Summary During the dormant period and especially during budbreak a poplar storage protein could be detected in xylem sap on SDS-PAGE and immunoblot; after budbreak it was absent. The permeability of poplar vessels and pit membranes for proteins larger than the poplar storage protein was confirmed, giving evidence for a long distance transport of entire storage proteins via the transpiration stream.
Key words: Populus, immunoblot, SDS-PAGE, storage protein, xylem sap. Abbreviation list: BSA = bovine serum albumin; PVDF dium dodecyl sulfate polyacrylamide gel electrophoresis.
=
Polyvinylidendifluorid; SDS-PAGE
--
Introduction The presence of sugars and amino acids in xylem sap is well established (cf. Sauter 1980, 1981, Tromp and Ovaa 1967). Mainly during budbreak they are carried to the buds in large amounts by means of the transpiration stream. In this paper the results of a search for proteins in xylem sap are reported. Materials and Methods
so-
=
66 kOa-
32 kOa-
Plant material Two-year-old twigs were collected from Populus x canadensis Moench «robusta» growing in the Botanical Garden of Kiel University.
Extraction of xylem sap and determination of vessel length Sap was sucked out of twig segments of 60 cm length with a vacuum pump (for special precautions see Sauter 1980). Prior to extraction the bark was removed from the twig and the cut surface was washed thoroughly in water to prevent contamination of xylem sap by proteins of injured cells. Part of the sap was filtered through a low protein binding PVDF membrane with a pore size of 0.22/lm to remove cell organells that might have polluted the sap. The vessel length was determined by sucking a dye suspension (magic color, french chartreuse Me 360, Royal Sovereign Graphics) through the twig. The particle size (about 1/lm) prevented them from penetrating the pit membranes and made them accumulate at the vessel ends. © 1991 by Gustav Fischer Verlag, Stuttgart
a
b
c
d
e
• f
Fig. 1: SDS-PAGE of unfiltered (a) and filtered (b) poplar xylem sap during budbreak showing the 32 kDa storage protein. Immunoblot of unfiltered (c) and filtered (d) xylem sap during budbreak. SDSPAGE of xylem sap in summer before addition of BSA (e) and after addition of BSA (f).
SDS·PAGE and immunoblotting Proteins of the xylem sap were precipitated in 80 % acetone, pelleted at 6000g (10 min), resolved in Laemmli buffer with 1 % mer-
764
BARBARA VAN CLEVE, JOACHIM JUST, and JORG J. SAUTER
captoethanol, separated by SDS-PAGE (Laemmli 1970) and transferred to nitrocellulose for immunoblotting (van Cleve et al. 1988). Results and discussion In winter and especially during budbreak a protein of about 32 kDa appeared in SDS-PAGE of poplar xylem sap, which was recognized by the antibody against poplar storage protein (Fig. 1 a, c). This protein vanished after budbreak (Fig. 1 e), as found for the storage protein of wood and bark (van Cleve et al. 1988, Wetzel et al. 1989 a, 1989b). SDS-PAGE and immunoblot of filtered and unfiltered sap showed the same amount of the 32kDa protein (Fig. 1 a-d); therefore a PVDF membrane with 0.221tm pores did not retain this protein. Since the protein bodies of poplar wood ray cells have a diameter of about 0.51tm (Sauter and Cleve 1989), they should be retained by the filter and thus cannot be the source of the protein in the xylem sap. Since every precaution was taken to prevent contamination of xylem sap by protein of damaged cells, i.e. by removal of bark, washing of cut surfaces and steril filtration of sap, the origin of the xylem sap protein from protein storing vacuoles of cut cells can be excluded. From these results we must conclude that the 32 kDa storage protein accumulated in protein bodies in ray cells (Sauter et al. 1988) is able to permeate into the vessels during the mobilization period in spring. However, there is still absolutely no information on how these protein molecules might pass from the living ray cell protoplast into the vessels.
In order to test the ability of protein to also pass the vessel end pit membrane of poplar, BSA, a much larger protein, i.e. 66 kDa, was sucked through a twig segment of 60 cm in length. Because no vessel was longer than 29 cm, the protein molecules had to pass the pit membranes of at least one vessel end. Sap was collected before and after addition of BSA. The SDS-PAGE of the collected xylem sap showed that the protein passed the vessel end pit membrane (Fig. 1 e-f). This experiment thus confirms that protein molecules of the size of the poplar storage protein (32 kDa) can permetrate the pit membranes of vessels. Therefore, a long distance transport not only of sugars and amino acids but also of storage protein molecules must be considered. Acknowledgements The skillful technical assistance of Miss Britta Marx is greatfully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft. References CLEVE, B. VAN, S. CLAUSEN, and J. J. SAUTER: J. Plant Physiol. 133, 371-374 (1988). LAEMMU, U. K.: Nature 227, 680-685 (1970). SAUTER, J. J.: Z. Pflanzenphysiol. 98, 377 - 391 (1980). - Z. Pflanzenphysiol. 101, 399-411 (1981). SAUTER, J. J., B. VAN CLEVE, and K. ApEL: Planta 173, 31-34 (1988). SAUTER, J. J. and B. VAN CLEVE: IAWA n.s., 395-403 (1989). TROMP, J. and J. C. OVAA: Z. Pflanzenphysiol. 57, 11-21 (1967). WETZEL, S., C. DEMMERS, andJ. S. GREENWOOD: Planta 178, 275-281 (1989 a). - - - Can. J. Bot. 67, 3439-3445 (1989 b).
Short Conlnlunication
Poplar Storage Protein in Xylem Sap BARBARA VAN CLEVE, JOACHIM JUST,
and JORG J.
SAUTER
Botanisches Institut der Universitat Kiel, OlshausenstraBe 40, D-2300 Kiel, Federal Republic of Germany Received November 7,1990 . Accepted November 27,1990
Summary During the dormant period and especially during budbreak a poplar storage protein could be detected in xylem sap on SDS-PAGE and immunoblot; after budbreak it was absent. The permeability of poplar vessels and pit membranes for proteins larger than the poplar storage protein was confirmed, giving evidence for a long distance transport of entire storage proteins via the transpiration stream.
Key words: Populus, immunoblot, SDS-PAGE, storage protein, xylem sap. Abbreviation list: BSA = bovine serum albumin; PVDF dium dodecyl sulfate polyacrylamide gel electrophoresis.
=
Polyvinylidendifluorid; SDS-PAGE
--
Introduction The presence of sugars and amino acids in xylem sap is well established (cf. Sauter 1980, 1981, Tromp and Ovaa 1967). Mainly during budbreak they are carried to the buds in large amounts by means of the transpiration stream. In this paper the results of a search for proteins in xylem sap are reported. Materials and Methods
so-
=
66 kOa-
32 kOa-
Plant material Two-year-old twigs were collected from Populus x canadensis Moench «robusta» growing in the Botanical Garden of Kiel University.
Extraction of xylem sap and determination of vessel length Sap was sucked out of twig segments of 60 cm length with a vacuum pump (for special precautions see Sauter 1980). Prior to extraction the bark was removed from the twig and the cut surface was washed thoroughly in water to prevent contamination of xylem sap by proteins of injured cells. Part of the sap was filtered through a low protein binding PVDF membrane with a pore size of 0.22/lm to remove cell organells that might have polluted the sap. The vessel length was determined by sucking a dye suspension (magic color, french chartreuse Me 360, Royal Sovereign Graphics) through the twig. The particle size (about 1/lm) prevented them from penetrating the pit membranes and made them accumulate at the vessel ends. © 1991 by Gustav Fischer Verlag, Stuttgart
a
b
c
d
e
• f
Fig. 1: SDS-PAGE of unfiltered (a) and filtered (b) poplar xylem sap during budbreak showing the 32 kDa storage protein. Immunoblot of unfiltered (c) and filtered (d) xylem sap during budbreak. SDSPAGE of xylem sap in summer before addition of BSA (e) and after addition of BSA (f).
SDS·PAGE and immunoblotting Proteins of the xylem sap were precipitated in 80 % acetone, pelleted at 6000g (10 min), resolved in Laemmli buffer with 1 % mer-
764
BARBARA VAN CLEVE, JOACHIM JUST, and JORG J. SAUTER
captoethanol, separated by SDS-PAGE (Laemmli 1970) and transferred to nitrocellulose for immunoblotting (van Cleve et al. 1988). Results and discussion In winter and especially during budbreak a protein of about 32 kDa appeared in SDS-PAGE of poplar xylem sap, which was recognized by the antibody against poplar storage protein (Fig. 1 a, c). This protein vanished after budbreak (Fig. 1 e), as found for the storage protein of wood and bark (van Cleve et al. 1988, Wetzel et al. 1989 a, 1989b). SDS-PAGE and immunoblot of filtered and unfiltered sap showed the same amount of the 32kDa protein (Fig. 1 a-d); therefore a PVDF membrane with 0.221tm pores did not retain this protein. Since the protein bodies of poplar wood ray cells have a diameter of about 0.51tm (Sauter and Cleve 1989), they should be retained by the filter and thus cannot be the source of the protein in the xylem sap. Since every precaution was taken to prevent contamination of xylem sap by protein of damaged cells, i.e. by removal of bark, washing of cut surfaces and steril filtration of sap, the origin of the xylem sap protein from protein storing vacuoles of cut cells can be excluded. From these results we must conclude that the 32 kDa storage protein accumulated in protein bodies in ray cells (Sauter et al. 1988) is able to permeate into the vessels during the mobilization period in spring. However, there is still absolutely no information on how these protein molecules might pass from the living ray cell protoplast into the vessels.
In order to test the ability of protein to also pass the vessel end pit membrane of poplar, BSA, a much larger protein, i.e. 66 kDa, was sucked through a twig segment of 60 cm in length. Because no vessel was longer than 29 cm, the protein molecules had to pass the pit membranes of at least one vessel end. Sap was collected before and after addition of BSA. The SDS-PAGE of the collected xylem sap showed that the protein passed the vessel end pit membrane (Fig. 1 e-f). This experiment thus confirms that protein molecules of the size of the poplar storage protein (32 kDa) can permetrate the pit membranes of vessels. Therefore, a long distance transport not only of sugars and amino acids but also of storage protein molecules must be considered. Acknowledgements The skillful technical assistance of Miss Britta Marx is greatfully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft. References CLEVE, B. VAN, S. CLAUSEN, and J. J. SAUTER: J. Plant Physiol. 133, 371-374 (1988). LAEMMU, U. K.: Nature 227, 680-685 (1970). SAUTER, J. J.: Z. Pflanzenphysiol. 98, 377 - 391 (1980). - Z. Pflanzenphysiol. 101, 399-411 (1981). SAUTER, J. J., B. VAN CLEVE, and K. ApEL: Planta 173, 31-34 (1988). SAUTER, J. J. and B. VAN CLEVE: IAWA n.s., 395-403 (1989). TROMP, J. and J. C. OVAA: Z. Pflanzenphysiol. 57, 11-21 (1967). WETZEL, S., C. DEMMERS, andJ. S. GREENWOOD: Planta 178, 275-281 (1989 a). - - - Can. J. Bot. 67, 3439-3445 (1989 b).