Planta

Planta 138, 279-294 (1978)

9 by Springer-Verlag 1978

Leaf Structure in Relation to Solute Transport and Phloem Loading in Zea mays L. R.F. Evert 1, W. Eschrich 2, and W. Heyser 2 Department of Botany, University of Wisconsin, Madison, WI 53706, USA and z Forstbotanisches Institut der Universitfit G6ttingen, Bfisgenweg 2, D-3400 G6ttingen-Weende, Federal Republic of Germany

Abstract. Small and intermediate (longitudinal) vascular bundles of the Zea mays leaf are surrounded by chlorenchymatous bundle sheaths and consist of one or two vessels, variable numbers of vascular parenchyma cells, and two or more sieve tubes some of which are associated with companion cells. Sieve tubes not associated with companion cells have relatively thick walls and commonly are in direct contact with the vessels. The thick-walled sieve tubes have abundant cytoplasmic connections with contiguous vascular parenchyma cells; in contrast, connections between vascular parenchyma cells and thin-walled sieve tubes are rare. Connections are abundant, however, between the thin-walled sieve tubes and their companion cells; the latter have few connections with the vascular parenchyma cells. Plasmolytic studies on leaves of plants taken directly from lighted growth chambers gave osmotic potential values of about - 1 8 bars for the companion cells and thin-walled sieve tubes (the companion cell-sieve tube complexes) and about - 11 bars for the vascular parenchyma cells. Judging from the distribution of connections between various cell types of the vascular bundles and from the osmotic potential values of those cell types, it appears that sugar is actively accumulated from the apoplast by the companion cell-sieve tube complex, probably across the plasmalemma of the companion cell. The thick-walled sieve tubes, with their close spatial association with the vessels and possession of plasmalemma tubules, may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream. The transverse veins have chlorenchymatous bundle sheaths and commonly contain a single vessel and sieve tube. Parenchymatic elements may or may not be present. Like the thick-walled sieve tubes of the longitudinal bundles, the sieve tubes of the transverse veins have plasmalemma tubules, indicating that they too may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream.

Key words: Apoplast - Companion cell-sieve tube complex - Leaf structure - Phloem loading - Sieve tubes - Symplast - Vascular bundle - Zea.

Introduction In a previous article (Evert et al., 1977a) we reported on the distribution and structure of the plasmodesmata between mesophyll, bundle-sheath, and vascular parenchyma cells in small and intermediate veins in the leaf of Zea mays L. Results of that study strongly indicated that, in the Z. mays leaf, movement of the photosynthetic intermediates between mesophyll and bundle-sheath cells is restricted largely or entirely to the plasmodesmata (symplastic pathway) while transpirational water movement is restricted largely or entirely to the cell walls (apoplastic pathway). The present study was undertaken primarily in an attempt to determine the probable pathway(s) of sugar from the bundle-sheath cells to the sieve tubes in small and intermediate bundles of the Z. mays leaf. To that end two sets of data were obtained: (1) the distribution and structure of the cytoplasmic connections between various cell types of the bundles, utilizing light, phase, and electron microscopy; and (2) the osmotic potentials of various cell types of the leaf by plasmolytic methods. The results demonstrate the great value structural information can bear on one's understanding of function in the plant body.

Materials and Methods Plant Material

The tissues used in this study were obtained from corn plants (Zea mays L., cv. Prior; Samen-Kr6bel, G6ttingen, Germany) grown

0032-0935/78/0138/0279/$03.20

280 in either the greenhouse (18-25~ natural daylight during summertime, additional mercury-vapor light, 16 h, during wintertime) or growth chambers (25~ humidity 65%, light 16 h, 3.10- 7 W . m -2) of the Forstbotanisches Institut, University of G6ttingen. Specifically, the tissue was obtained from the median portions of the 5th visible leaf, counted from above, of plants ca. 80 cm tall.

R.F. Evert et al. : Leaf Structure and Phloem Transport in Zea mounted in distilled water. Plasmolysis was determined as that point in the increasing concentration series at which approximately 50% or more of the cells of a given type exhibited plasmolysis after 2 h. The sucrose solutions ranged from 0.1 to 1.5 M in steps of 0.1 M.

Results Structural Studies

Small and Intermediate Bundles Some tissues were fixed in 6% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.0, for 6 h at r o o m temperature and post-fixed in 2% o s m i u m tetroxidein 0.05 M cacodylate buffer overnight in a refrigerator; others were fixed solely in 2% o s m i u m tetroxide. With both procedures tissues were obtained both from the leaves of plants taken directly from lighted growth chambers ( " l i g h t " plants) and from the leaves of growth-chamber-grown plants that had been placed in the dark for 4 8 h prior to fixation ( " d a r k " plants). Embedment was in either Epon-Araldite (Ted Pella, Tustin, Cal., USA) or Spurr's Epoxy resin (Spurr, 1969). 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. In addition to examination of a great many vascular bundles in thin sections, 351 free-hand drawings from as m a n y sections were made of 11 small to intermediate vascular bundles, as seen with the electron microscope. The distribution and structure of connections between different cell types and of various cellular components within those cell types were recorded on the drawings. Investigation of the tissues with the electron microscope was complemented with bright-field and phase-contrast microscope examination of both macerated tissues and free-hand sections of living tissues.

Plasrnolytic Studies Plasmolytic studies were conducted directly on living tissues with the light microscope. These studies, like the structural ones, were carried out on leaves of plants taken directly from lighted growth chambers, and on leaves of growth-chamber-grown plants that had been placed in the dark for 48h ( " l i g h t " and " d a r k " plants1). Also as with the structural studies, the plasmolytic studies were conducted on tissues obtained from median portions of the 5th visible leaf, counted from above. Portions of the blade 3.5cm in length were removed from the leaves with sharp razor blades, placed directly in tap water in a flask, and gently aspirated. Flasks containing " d a r k " tissues were covered with a l u m i n u m foil. Sharp razor blades were used to obtain longitudinal sections ca. 0.5 m m x 20 m m from median portions of the 3.5-cm blade segments. During this procedure the leaf segments and sections were continually immersed in water. As quickly as possible, the leaf sections were mounted on slides in graded sucrose solutions and cover slips were applied. The preparations were studied immediately with a light microscope and compared with control sections The initial plasmolytic studies were carried out on leaf tissues obtained from greenhouse-grown plants, These studies were however discontinued when it was found that the osmotic potential of various cell types within the leaf varied greatly from day to day, making it impossible to obtain consistent experimental results with greenhouse-grown plants over a period of several days. Higher osmotic potentials were encountered on clear days than on cloudy ones. Results obtained with growth-chamber-grown plants were fairly uniform from one day to the next, and it is those results that are reported herein

Brief Description. Both small and intermediate vascular bundles of the Z. mays leaf have chlorenchymatons bundle sheaths, the outer tangential and radial cell wall of which exhibit a continuous suberin lamella (Evert et al., 1977a). The so-called small bundles intergrade in size with those designated as intermediate, the latter sometimes differing from some small bundles primarily by the presence of a hypodermal sclerenchyma strand that commonly occurs on both sides of the bundle between epidermis and sheath (see Fig. 1, Evert etal., 1977a). The vascular tissues of the small bundles mature after elongation of the leaf, and by definition are metaxylem and metaphloem (Esau, 1943). With apparently few exceptions, the intermediate bundles also consist entirely of metaxylem and metaphloem. In most bundles the xylem contains one or two vessels commonly bordered by one or more files of parenchyma cells, which often are also in contact with phloem cells (Figs. 1, 3, 4, 15). These and other parenchyma cells of the bundles typically contain plastids with electron-dense protein crystalloids and are referred to by the general designation, vascular parenchyma cells. In intermediate bundles and some small ones, the plastids of some parenchyma cells may not contain crystalloids. Such cells, which resemble the vascular parenchyma cells in other respects,

Figs. 1 and 2. Transverse sections of portions of a small vascular bundle from leaf of a Zea rnays plant kept in the dark for 48 h. Fixation in glutaraldehyde and postfixation in OsO~. BS, bundle sheath; CC, c o m p a n i o n cell; 1S, intercellular space; ST, sieve tube; V, vessel; VP, vascular parenchyma cell Fig. 1. This smalt bundle contains only two sieve tubes, a thickwalled one contiguous to the vessel and a thin-walled one, below. In bundles fiom " d a r k " plants the thin-walled sieve tubes and their companion cells were plasmolyzed when fixed in glutaraldehyde. The bundle-sheath chloroplasts of all " d a r k " plants lacked starch grains, x4,800; b a r = 2 . 1 0 tam Fig.2. Detail of Figure l showing portion of wall between vascular parenchyma cell (below) and vessel (above) at site where secondary thickening of vessel is lacking. Only remnants of the primary wall of the vessel are discernible (arrows). The arrowhead points to hydrolyzed primary wall between vessel members, x27,900; b a r = 0 . 3 6 gm

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Leaf structure in relation to solute transport and phloem loading in Zea mays L.

Small and intermediate (longitudinal) vascular bundles of the Zea mays leaf are surrounded by chlorenchymatous bundle sheaths and consist of one or tw...
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