Protoplasma (1992) 166: 153-164 9 Springer-Verlag 1992 Printed in Austria Living sieve cells of conifers as visualized by confocal, laser-scanning fluorescence microscopy A. Schulz* Zellenlehre, Universit/it Heidelberg, Heidelberg Received April 8, 1991 Accepted July 5, 1991 Summary. Confocal laser scanning microscopy (CLSM) and fluo- rochromes were used to visualize the assimilate-conducting sieve cells of conifers in vivo. When still nucleate, the cytoplasm of these cells shows streaming and occupies the cell periphery including the pit- like, thin watl regions where sieve areas would develop. During differentiation the nuclear fluorescence and the central vacuoles dis- appear. At maturity and after ER-specific staining the sieve areas are the most conspicuous character of sieve cells, Those linking two sieve cells are covered on either side with prominent amounts of ER, while those leading to a Strasburger (= albuminous) cell show flu- orescence on the sieve-cell side only. Within the sieve-area wall flu- orescence appears also in the common median cavity which is part of the symplastic path between sieve ceils. Electron microscopy (EM) depicts the ER as complexes of densely convoluted tubules of smooth ER, equally on either side of a sieve area, provided that the fixation of this sensitive tissue is appropriate. Purposeful wounding causes a swelling and vesiculation of the ER-tubules which is visible in both CLSM and EM. Electron micrographs of ER-complexes at sieve areas - in this paper demonstrated in vivo - have often been argued to be artefacts, since they should raise flow resistance considerably and are not consistent with the Miinch hypothesis on phloem trans- port. The implication,~ of this location for phloem transport are discussed. Keywor~: Conifers; Endoplasmic reticulum; Laser scanning mi- croscopy; Phloem transport; Sieve areas. Abbreviations: CLSM confocal laser scanning microscopy; DiOC 3,3'-dioxacarbocyanine iodide; EM electron microscopy; ER en- doplasmic reticnlum; FDA fluorescein diaeetate. Introduction In vascular plants the translocation ofphotoassimilates from source to sink takes place in the sieve elements. * Correspondence and reprints: Botanisches Institut, Universitfit Kiel, Olshausenstrasse 40, D-W-2300 Kiel 1, Federal Republic of Germany. These most specialized cells of the phloem are contin- uous throughout the plant body and are particularly well connected by sieve pores. The relation between structure and function of the sieve elements is not yet fully understood. According to Milburn and Kallar- ackal (1989) the most widely accepted mechanism for phloem transport is the pressure-driven mass flow as postulated by Mtinch (1930). This hypothesis requires sieve elements with minimum resistance from static structures, as was pointed out by Weatherley (t975 a). The degree of resistance depends upon the number and width of sieve pores, and upon the sieve-element cy- toplasm. When sieve elements reach maturity, their nucleus and most organelles are degenerated or have disappeared. Apart from parietal plastids and mito- chondria, however, sieve elements of angiosperms pre- serve prominent amounts of P-protein, and those of gymnosperms extended, network-like complexes of the ER as seen in electron microscopy (for review, see Behnke and Sjolund 1990). These compounds should raise flow resistance considerably when present in the cell lumen and even more when covering or occluding the sieve pores as electron micrographs often suggested. This technique, however, provides only static images and is moreover subject to artefacts introduced by the preparation and fixation procedure itself. In order to avoid fixation artefacts, the present paper approaches the in vivo localization of ER-complexes in sieve ele- ments of a conifer with confocal microscopy and vital dyes. For tissue preparation the bark, including up to five year-old secondary phloem, was only stripped from the wood, infiltrated with dyes and observed without