Planta 135,203
205(1977)
P l a n t a 9 by Springer-Verlag 1977
Short Communication
Tubular Extensions of the Plasmalemma in Leaf Cells of Zea mays L. R.F. Evert, W. Eschrich, D.S. Neuberger, and S.E. Eichhorn Department of Botany, University of Wisconsin, Madison, WI 53706, USA and Forstbotanisches Institut der Universitfit G6ttingen, B/isgenweg 2, D-3400 G6ttingen-Weende, Federal Republic of Germany
Abstract. Leaf tissues of Zea mays were examined with a transmission electron microscope and a highvoltage electron microscope. Tubular extensions (invaginations) of the plasmalemma were found in vascular parenchyma cells and thick-walled, lateformed sieve elements of intermediate and small veins, and in epidermal, mesophyll, and sheath cells of all leaves examined. No continuity seems to exist between the tubules and other cellular membranes. Key words: Apoplast - Leaf cells - Plasmalemma extensions - Symplast - Zea.
During the past few years interest in the pathways for the short-distance transfer of substances in plants, especially in leaves, has greatly increased (Osmond and Smith, 1976; and literature cited therein). The pathways for both the intermediates and the products of photosynthesis and the solutes moving from the xylem to other leaf tissues, may be symplastic or apoplastic or a combination of both. At this time we are reporting on the presence of tubular extensions (invaginations) of the plasmalemma in leaf cells of Zea mays L., the leaves of which have been the object of detailed examination in our laboratories with both light and electron microscopes. The tubules apparently greatly extend the surface of the plasmalemma and concomitantly increase the apoplast-symplast interface. Some of the tissues used in this study were obtained from corn plants (Zea mays L., cv. Prior) grown in either the greenhouse or growth chambers of the Forstbotanisches Institut, University of G6ttingen; others were obtained from corn plants (W117 Texas cytoplasm-male sterile; University of Wisconsin) grown in growth chambers of the Department of Plant Pathology, University of Wisconsin-Madison. Some tissues were initially fixed in glutaraldehyde and postfixed in OsO4, others were fixed soleIy in OsO4. Embedment was in either epon-araldite or Spurr's epoxy resin.
Thin sections were cut with a diamond knife on a Porter-Blum MT-2 ultramicrotome, stained with uranyl acetate and lead citrate, and viewed and photographed with a Hitachi HU-11C microscope. Sections 0.5 gm thick, stained with uranyl magnesium acetate and lead citrate, were viewed and photographed at 1 MeV with the High-Voltage Electron Microscope (HVEM) at the University of Wisconsin.
Tubular extensions of the plasmalemma were encountered in vascular parenchyma cells (Fig. l) and the thick-walled, late-formed sieve elements (Fig. 2) of small and intermediate veins, and in epidermal, mesophyll (Fig. 3), and sheath (Fig. 4) cells of all leaves examined. (Large veins were not examined in detail.) Most of the extensions arose near the plasmodesmata of the parenchymatic elements (Figs. 1, 3) and near pores of the plasmodesmata-sieve pore connections joining the protoplast of vascular parenchyma cells with those of the thick-walled sieve elements (Fig. 2). The tubules are less conspicuous in tissues fixed in glutaraldehyde and postfixed in OsO4 than in OsO4-fixed tissues. In the latter the outer layer of the tripartite plasmalemma often appears thicker and more heavily stained than the inner one (Figs. 3-5). Consequently, the tubular extensions also are asymmetric in appearance but with the darker, thicker layer on the inside. This appearance facilitates the identification of isolated portions of the tubules. The outer dimensions of most tubules measured about 0.8 nm in diameter. Portions of some tubules are dilated (Figs. 2, 5). Tubules that may have been derived from the plasmalemma have been reported in plant cells infected with viruses (Gibbs, 1976). However, the tubular extensions of the plasmalemma were found in the same cell types of definitely virus-free material (Wl17 Texas, provided by G.A. de Zoeten) as in those of the Prior plants which, although apparently healthy, may have been virus-infected. Consequently, we con-
204
R.F. Evert et al. : Plasmalemma Extensions in Zea Leaf Cells
R.F. Evert et al. : Plasmalemma Extensions in Zea Leaf Cells
Figs. 1-6. Transmission electron micrographs. All from tissues fixed solely in OsO4. Unlabeled arrows point to sites of origin of tubular extensions of plasmalemma, In all figures, C callose, CP chloroplast, ER endoplasmic reticulum, PD plasmodesma, S suberized wall of sheath cell, T tubular extensions of plasmalemma, V vacuole Fig. 1. Portions of two vascular parenchyma cells with tubular extensions of plasmalemma in parietal layer of cytoplasm and in cytoplasmic strands (upper cell) opposite a group of plasmodesmata. x 62,400; bar = 0.16 gm Fig. 2. Portions of mature, late-formed sieve element (right) and vascular parenchyma cell (left) with tubular extensions of plasmalemma near or opposite pores in the sieve element. The pores shown here are almost occluded by callose. Parts of tubules may become dilated. • 61,200; bar=0.16 gm Fig. 3. Portions of mesophyll (left) and sheath (right) cells with tubular extensions of plasmalemma, x 62,400 ; bar = 0.16 gm Fig. 4. Portions of mesophyll (left) and sheath (right) cells showing transverse, oblique and longitudinal profiles of tubular extensions of plasmalemma in the sheath ceil. Note asymmetrical appearance of the tripartite plasmalemma, x 59,200; bar=0.17 gm Fig. 5, Tubular extensions of plasmalemma in a mesophyll cell. Part of one tubule is dilated. Note asymmetrical appearance of the tubule membrane. • 67,000; bar=0.15 gm Fig. 6. Portion of chloroplast from mesophyll. The two tubular extensions of the plasmalemma shown here appear to unite with the plastid envelope. However, actual continuities between the tubules and other cellular components could not be confirmed. • 62,000; bar=0.I6 gm
clude that the tubules are normal components of the Zea leaf cells. While continuity has been reported between the plasmalemma and the membranes of other organelles (endoplasmic reticulum, nuclear envelope, mitochondria, and chloroplasts), no continuity seems to exist between the tubular extensions of the plasmalemma and other cell membranes in the leaf of Zea. Kursanov
205
and Paramonova (1976) reported that in "actively functioning," mature sugar-beet leaves the plasmalemma forms numerous folds, some of which establish structural continuity with the outer chloroplast membrane. They emphasized the significance of such contacts as a means of facilitating "evacuation and admission of substances" directly from the chloroplast into the free space and back. Although some tubules appeared to be continuous with the outer membranes of chloroplasts in thin sections with the transmission electron microscope (Fig. 6), this was in no case confirmed by examination of pairs of stereo micrographs obtained with the HVEM. Nevertheless, the tubules apparently greatly extend the surface of the plasmalemma. This work was initiated in 1975, while the first author was an Alexander von Humboldt-Stiftung Awardee at the Forstbotanisches Institut G6ttingen. Thanks are extended to Lynn Smook and Mary Binkley for their technical assistance. This research was supported in part by National Science Foundation grant DEB7301624 to R. F. E. The AEI, EM-7 high-voltage electron microscope is supported by National Institutes of Health research grant RR00570 from the Biotechnology Resources Branch, Division of Research Resources.
References Gibbs, A. : Viruses and plasmodesmata. In : Intercellular communication in plants: Studies on plasmodesmata, pp. 149-164, Gunning, B.E.S., Robards, A.W., eds. Berlin-HeidelbergNew York: Springer 1976 Kursanov, A.L., Paramonova, N.V.: Ultrastructural changes in the mesophyll of leaves of Beta vulgaris L. in connection with assimilate transport. Soviet Plant Physiol. 23, 242-248 (1976) Osmond, C.B., Smith, F.A.: Symplastic transport of metabolites during C4-photosynthesis. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 229-240, Gunning, B.E.S., Robards, A.W., eds. Berlin-Heidelberg-New York: Springer 1976
Received 14 February; accepted 14 March 1977
205(1977)
P l a n t a 9 by Springer-Verlag 1977
Short Communication
Tubular Extensions of the Plasmalemma in Leaf Cells of Zea mays L. R.F. Evert, W. Eschrich, D.S. Neuberger, and S.E. Eichhorn Department of Botany, University of Wisconsin, Madison, WI 53706, USA and Forstbotanisches Institut der Universitfit G6ttingen, B/isgenweg 2, D-3400 G6ttingen-Weende, Federal Republic of Germany
Abstract. Leaf tissues of Zea mays were examined with a transmission electron microscope and a highvoltage electron microscope. Tubular extensions (invaginations) of the plasmalemma were found in vascular parenchyma cells and thick-walled, lateformed sieve elements of intermediate and small veins, and in epidermal, mesophyll, and sheath cells of all leaves examined. No continuity seems to exist between the tubules and other cellular membranes. Key words: Apoplast - Leaf cells - Plasmalemma extensions - Symplast - Zea.
During the past few years interest in the pathways for the short-distance transfer of substances in plants, especially in leaves, has greatly increased (Osmond and Smith, 1976; and literature cited therein). The pathways for both the intermediates and the products of photosynthesis and the solutes moving from the xylem to other leaf tissues, may be symplastic or apoplastic or a combination of both. At this time we are reporting on the presence of tubular extensions (invaginations) of the plasmalemma in leaf cells of Zea mays L., the leaves of which have been the object of detailed examination in our laboratories with both light and electron microscopes. The tubules apparently greatly extend the surface of the plasmalemma and concomitantly increase the apoplast-symplast interface. Some of the tissues used in this study were obtained from corn plants (Zea mays L., cv. Prior) grown in either the greenhouse or growth chambers of the Forstbotanisches Institut, University of G6ttingen; others were obtained from corn plants (W117 Texas cytoplasm-male sterile; University of Wisconsin) grown in growth chambers of the Department of Plant Pathology, University of Wisconsin-Madison. Some tissues were initially fixed in glutaraldehyde and postfixed in OsO4, others were fixed soleIy in OsO4. Embedment was in either epon-araldite or Spurr's epoxy resin.
Thin sections were cut with a diamond knife on a Porter-Blum MT-2 ultramicrotome, stained with uranyl acetate and lead citrate, and viewed and photographed with a Hitachi HU-11C microscope. Sections 0.5 gm thick, stained with uranyl magnesium acetate and lead citrate, were viewed and photographed at 1 MeV with the High-Voltage Electron Microscope (HVEM) at the University of Wisconsin.
Tubular extensions of the plasmalemma were encountered in vascular parenchyma cells (Fig. l) and the thick-walled, late-formed sieve elements (Fig. 2) of small and intermediate veins, and in epidermal, mesophyll (Fig. 3), and sheath (Fig. 4) cells of all leaves examined. (Large veins were not examined in detail.) Most of the extensions arose near the plasmodesmata of the parenchymatic elements (Figs. 1, 3) and near pores of the plasmodesmata-sieve pore connections joining the protoplast of vascular parenchyma cells with those of the thick-walled sieve elements (Fig. 2). The tubules are less conspicuous in tissues fixed in glutaraldehyde and postfixed in OsO4 than in OsO4-fixed tissues. In the latter the outer layer of the tripartite plasmalemma often appears thicker and more heavily stained than the inner one (Figs. 3-5). Consequently, the tubular extensions also are asymmetric in appearance but with the darker, thicker layer on the inside. This appearance facilitates the identification of isolated portions of the tubules. The outer dimensions of most tubules measured about 0.8 nm in diameter. Portions of some tubules are dilated (Figs. 2, 5). Tubules that may have been derived from the plasmalemma have been reported in plant cells infected with viruses (Gibbs, 1976). However, the tubular extensions of the plasmalemma were found in the same cell types of definitely virus-free material (Wl17 Texas, provided by G.A. de Zoeten) as in those of the Prior plants which, although apparently healthy, may have been virus-infected. Consequently, we con-
204
R.F. Evert et al. : Plasmalemma Extensions in Zea Leaf Cells
R.F. Evert et al. : Plasmalemma Extensions in Zea Leaf Cells
Figs. 1-6. Transmission electron micrographs. All from tissues fixed solely in OsO4. Unlabeled arrows point to sites of origin of tubular extensions of plasmalemma, In all figures, C callose, CP chloroplast, ER endoplasmic reticulum, PD plasmodesma, S suberized wall of sheath cell, T tubular extensions of plasmalemma, V vacuole Fig. 1. Portions of two vascular parenchyma cells with tubular extensions of plasmalemma in parietal layer of cytoplasm and in cytoplasmic strands (upper cell) opposite a group of plasmodesmata. x 62,400; bar = 0.16 gm Fig. 2. Portions of mature, late-formed sieve element (right) and vascular parenchyma cell (left) with tubular extensions of plasmalemma near or opposite pores in the sieve element. The pores shown here are almost occluded by callose. Parts of tubules may become dilated. • 61,200; bar=0.16 gm Fig. 3. Portions of mesophyll (left) and sheath (right) cells with tubular extensions of plasmalemma, x 62,400 ; bar = 0.16 gm Fig. 4. Portions of mesophyll (left) and sheath (right) cells showing transverse, oblique and longitudinal profiles of tubular extensions of plasmalemma in the sheath ceil. Note asymmetrical appearance of the tripartite plasmalemma, x 59,200; bar=0.17 gm Fig. 5, Tubular extensions of plasmalemma in a mesophyll cell. Part of one tubule is dilated. Note asymmetrical appearance of the tubule membrane. • 67,000; bar=0.15 gm Fig. 6. Portion of chloroplast from mesophyll. The two tubular extensions of the plasmalemma shown here appear to unite with the plastid envelope. However, actual continuities between the tubules and other cellular components could not be confirmed. • 62,000; bar=0.I6 gm
clude that the tubules are normal components of the Zea leaf cells. While continuity has been reported between the plasmalemma and the membranes of other organelles (endoplasmic reticulum, nuclear envelope, mitochondria, and chloroplasts), no continuity seems to exist between the tubular extensions of the plasmalemma and other cell membranes in the leaf of Zea. Kursanov
205
and Paramonova (1976) reported that in "actively functioning," mature sugar-beet leaves the plasmalemma forms numerous folds, some of which establish structural continuity with the outer chloroplast membrane. They emphasized the significance of such contacts as a means of facilitating "evacuation and admission of substances" directly from the chloroplast into the free space and back. Although some tubules appeared to be continuous with the outer membranes of chloroplasts in thin sections with the transmission electron microscope (Fig. 6), this was in no case confirmed by examination of pairs of stereo micrographs obtained with the HVEM. Nevertheless, the tubules apparently greatly extend the surface of the plasmalemma. This work was initiated in 1975, while the first author was an Alexander von Humboldt-Stiftung Awardee at the Forstbotanisches Institut G6ttingen. Thanks are extended to Lynn Smook and Mary Binkley for their technical assistance. This research was supported in part by National Science Foundation grant DEB7301624 to R. F. E. The AEI, EM-7 high-voltage electron microscope is supported by National Institutes of Health research grant RR00570 from the Biotechnology Resources Branch, Division of Research Resources.
References Gibbs, A. : Viruses and plasmodesmata. In : Intercellular communication in plants: Studies on plasmodesmata, pp. 149-164, Gunning, B.E.S., Robards, A.W., eds. Berlin-HeidelbergNew York: Springer 1976 Kursanov, A.L., Paramonova, N.V.: Ultrastructural changes in the mesophyll of leaves of Beta vulgaris L. in connection with assimilate transport. Soviet Plant Physiol. 23, 242-248 (1976) Osmond, C.B., Smith, F.A.: Symplastic transport of metabolites during C4-photosynthesis. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 229-240, Gunning, B.E.S., Robards, A.W., eds. Berlin-Heidelberg-New York: Springer 1976
Received 14 February; accepted 14 March 1977
