living sieve cells of conifers as visualized by confocal, laser-scanning fluorescence microscopy

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 fluorescence on the sieve-cell side only. Within the sieve-area wall fluorescence 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 transport. The implication,~ of this location for phloem transport are discussed.

Keywor~: Conifers; Endoplasmic reticulum; Laser scanning microscopy; Phloem transport; Sieve areas.

Abbreviations: CLSM confocal laser scanning microscopy; DiOC 3,3'-dioxacarbocyanine iodide; EM electron microscopy; ER endoplasmic 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 continuous 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 cytoplasm. When sieve elements reach maturity, their nucleus and most organelles are degenerated or have disappeared. Apart from parietal plastids and mitochondria, 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 elements 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

154 A. Schutz: Living sieve cells of conifers

fur ther cu t t ing o f the sample. Star t ing at the exposed

c a m b i u m , the chosen ins ide-out -v iew m a d e deve lop ing

and m a t u r e sieve cells accessible for mic roscopy . Con-

focal laser scanning f luorescence mic roscopy al lows the

obse rva t ion o f opt ica l sect ions o f 1.2 ~tm thickness in-

dependen t o f specimen thickness (see W h i t e e t a l . 1987,

Schulz 1988, Sho t t on 1989). In o rde r to evalua te ar-

t ificial changes, those sieve areas were also viewed

which, close to a cut, showed w o u n d responses.

Materials and methods

Plant material

Secondary phloem of spruces (Picea abies (L.) Karst.) was collected (t) for live observation with the laser scanning microscope (CLSM) from a tree in the surrounding greenland of the department in Au- gust, and (2) for electron microscopic fixations from trees at the "K/ilbelescheuer" in the Blackforest in November.

Preparation for live observation

Branches were cut from the tree and kept with the cut end in water, at most 30 rain before microscopy. Longitudinal bark pieces of 4 by 1 cm were cut out of the 3- to 5-year-old branch region with a knife. The pieces were peeled off with tweezers so that the phloem was separated from the xylem. To avoid further wound artefacts due to preparatory cuts, the bark pieces were placed entirely into the vital dyes for 15 rain. Fluorescein diaeetate (FDA) was used in order to test cell viability (Heslop- Harrison and Heslop-Harrison 1970) and to observe organelle movement (SchuIz I988). From the stock solution (5mg FDA in l ml acetone) 50 gl were diluted in 5 ml H20. 3,3"-dioxacarbocyanine iodide (DiOC; Quader and Schnepf 1986) was dissolved in dimethylsulfoxide, from which 50 gl were diluted in 5 ml H20. After several washes in water, the bark pieces were placed cambium- up on a 76 by 26 mm cover slip (0.I7 mm thick; as used with inverted microscopes), mounted in water and covered with a second cover slip. This sandwich was fixed by springs in a way that the lower slide was bent and the upper one formed a plane ready for immersion of the objective lens (Fig. I).

ConfocaI laser scanning fluorescence microscopy ( CLSM)

For fluorescence micrographs a CLSM (Heidelberg Instruments, now Leica Laser Technik, Heidelberg) with epi-illumination was used in confocal mode, except for Fig. 2 (conventional microscope) and Fig. 4 (CLSM in non-confocal mode). By the confocal set-up of the apertures (= pin-holes) responsible for the exciting laser beam and the photomultiplier-enhanced fluorescence detection, respectively, fluorescence signals are restricted to the actual optical section of 1.2 lJm thickness. By raising and lowering the stage, the optical section can be moved vertically through intact specimens. For FDA and DiOC, the wave length of the exciting laser source was 488 rim, and a beam splitter of 492nm, and a barrier filter of 515nm wave length were used.

Electron microscopy (Elff )

For comparison of the living sieve cells with those fixed for electron microscopy (Figs. 15-18), representative samples were chosen from

Fig. 1. Mounting device for confocal 14laser scanning microscopy (CLSM) of entire bark portions. Thin object slides with the specimen (B) in between were held together by springs in a way that the upper one formed a plane, the lower one was bent. The gap in between them was filled with water; both ends of the bark were kept wet with cotton wool

a series that was originally collected over two growing seasons for a forest-dieback research project (see Schulz and Behnke 1987). Lon- gitudinal bark pieces were immersed into Karnovsky-fixation mix- ture (Karnovsky 1965) immediately at the location. Before further procession in the lab, tangential hand sections were taken so as to separate the functional secondary phloem from older bark regions.

Results

Tangent ia l sections o f func t iona l secondary ph loem of

P inaceae show n u m e r o u s th ick-wal led sieve cells in-

te rspersed by axia l and rad ia l p a r e n c h y m a cells (see

Schulz 1990). W i t h bluel ight exc i ta t ion and the chosen

filter combina t ion , au tof luorescence appea r s only in

axial p a r e n c h y m a cells wi th t ann in vacuoles. Fo l l owing

s ta ining wi th D i O C and conven t iona l f luorescence mi-

c roscopy, the sieve cells are recognized by f luorescent

sieve areas in their rad ia l cell walls (Fig. 2).

C L S M o f an ent ire b a r k piece in tangent ia l view reveals

much be t te r con t r a s t (Fig. 3). The fa in t ly f luorescent

lumina o f the sieve cells and the d a r k secondary cell

walls are clear ly d iscr iminated . The re la t ion o f length

to wid th o f the sieve cells is in the range f rom 50 : 1 to

100 : 1. Therefore , the long- t ape r ing end walls a re only

rare ly exhibi ted. They bear a series o f sieve areas each

charac te r ized by pai red , f luorescence-posi t ive spots

(Fig. 3). In func t iona l ph loem, only p a r e n c h y m a cells

con ta in nuclei, the envelope o f which is s ta ined with

D i O C (Fig. 3).

The effect o f the confocal m o d e o f the C L S M is dem-

ons t ra ted in a t ransverse sect ion o f a resin duct. F o r

Fig. 4 the ape r tu re a t the f luorescence-detect ing pho -

tomul t ip l i e r is removed . A l t h o u g h con t r a s t is still be t te r

than in conven t iona l mic roscopy , r e so lu t ion suffers

f rom b lur r ing due to f luorescence planes f rom o ther

than the ac tua l image plane. In the confoca l m o d e

(detec t ion ape r tu re effective), the ray cells have a

DiOC-pos i t ive , par t i cu la te c y t o p l a s m and non-f lu-

orescent walls and vacuoles (Fig. 5).

Figs. 2-5. Comparison of conventional and confocal fluorescence microscopy of mature secondary phloem of spruce stained with DiOC (tangential view)

Fig. 2. The conventional epifluorescence micrograph of a hand section shows parallel sieve cells (SC), recognized by fluorescent sieve areas (arrows) in the thick cell walls (w), and a ray with a nucleate (n) parenchyma cell. Bar: 10 gm

Fig. 3. In form of an opticaI section of 1.2 gm thickness, CLSM produces a much more detailed image of a similar tissue portion. Non- fluorescent walls (w,) bear sieve areas (arrows) that are densely distributed over an end wall between two sieve cells (between arrowheads). The Iurnen of the sieve cells (SC) contains numerous fluorescent organelles, but a nucleus is not visible, n Nucleus of a ray celt. Bar: i0 gm

Fig. 4. Cross section of a ray visualized in non-confocal mode of the CLSM with blurring fluorescence of parenchyma cells (PC) that surround a resin duct. Bar: 20 gm

Fig. 5. In confocal mode the same cells are seen in a 1.2 gm thick section. The non-fluorescent vacuoles (v)and cell walls are clearly discriminated from the organelles-containing cytoplasm of the ray cells, rd Resin duct. Bar: 20 gm

156 A. Schulz: Living sieve cells of conifers

Developing sieve cells

The developing phloem inside the mature region (closer to the cambium, Figs. 6-8) has still nucleate sieve cells. In this stage they are able to split the (non-fluorescent) dye FDA into the fluorescing compound fluorcscein and acetic acid. This reaction between endogenous es- terases and FDA is normally used to test the viability of pollen grains, suspension culture cells and proto- plasts (see Heslop-Harrison and Heslop-Harrison 1970, Widholm 1972). In young sieve cells the nuclei accumulate more fluorescein than the parietal cytoplasm (Fig. 7). Cell wall and the vacuoles are not stained (Figs. 6 and 7). In higher magnification spherical organelles appear that, according to their size and fre- quency, presumably are mitochondria and/or plastids (Fig. 7). Sieve areas are dent-like depressions of the walls. CLSM allows the summing of successive scans in order to reduce noise from the photomuitiplier. One scan takes approx. 1 s. If ten scans are summed up, any organelle movement within the cytoplasm should cause distortions. Such distortions are depicted in Fig. 7 A, lower sieve cell. Organelles seem to move parallel, since their spherical shapes are distorted to parallel ellipses. In the following image summed from 10 scans (Fig. 7 B) most of them have disappeared out of the image plane. In contrast to the general fluorescence of the cytoplasm after FDA staining, DiOC gives rise to fluorescence limited to the nuclear and plastid envelopes, the entire mitochondria and the cndomembrane system. A portion of immature phloem is shown in Fig. 8. Because of the large extensions of conifer sieve cells, nuclei are only rarely found. When they were, they often showed a lobing of their outline in contrast to the spherical nuclei in parenchyma cells (as, e.g., in Fig. 9). The central lumen of a sieve cell contains large, elongated vac-

l~gs. 6 and 7. CLSM of immature phloem stained with FDA

Fig. 6. Only living cells are able to accumulate the fluorescent compound of the dye. They contain nuclei (n) and a large vacuolar space (v) in the central lumen. Future sieve areas are anticipated by dent- like depressions (arrowheads) of the sieve-cell walls and filled with fluorescent cytoplasm, Bar: 10tim

Fig. 7. A The nucleus (n) and spherical organelles (arrows) accumulate more fluorescein than the ground plasm. In the upper sieve cell these organelles were stationary over 10 scans of 1 second each. In the lower the organelles were moving, thus being distorted to elongated forms, v Vacuoles, w cell wall B Within the next set of 10 scans the organelles of the lower sieve cell have disappeared out of the optical plane. Bars: 10gm

Fig.8. CLSM of immature phloem stained with DiOC. Developing sieve cells (SC) with a lobed nucleus (n), vacuoles (v), and pit-like depressions (arrows) where sieve areas would develop. The parietal cytoplasm contains spherical organdies (small arrowheads). Bar: 10 gm

Fig. 9. In mature phloem, the sieve areas are characterized by ER-comptexes on either side when linking sieve cells (arrows), and on the sieve- cell side only, when leading to a Strasburger cell (arrowheads). n Nucleus of a ray parenchyma cell, SC sieve cell, StC Strasburger cell, w cell wall. Bar: I0 gm


Figs. 10-12. Sieve areas stained with DiOC in 3 D-analysis

Fig. 10A, B. Two optical sections out of a series depicting three sieve areas in XY-planes (laser beam was scanned in X- and Y-axis). A The positions of Figs. 1 t A -G are indicated which as XZ-planes are oriented perpendicular to Fig. 10. SC Sieve-cell lumen, w cell watt. Bar: 10 pm

Fig. 11 A-G. CLSM produces XZ-images by scanning the laser beam in X-axis and step-wise moving the table. These images start in the upper sieve cell of Fig. 10A and move through the common wail (C and D) into the lower one. In this face view the ER complexes appear

as elliptic discs, interrupted by non-fluorescent organelles. • 1,200

Fig. 12. Higher magnification of Fig. 10 makes the fluorescing envelope of plastids (p), and the median cavity in the region of the middle lamella (small arrowheads) visible, er ER-complexes, Bar: 10 ~tm

Figs, 13-18. Comparison of CLSM of sieve areas in vivo (Figs. 13 and 14) and after prolonged fixation (Figs. 17 and 18) with EM-micrographs

(Figs. 15 and 16)

Fig. 13. In vivo, the ER-complexes (er) occur on either side of a sieve area. w Cell wall; small arrowheads, fluorescence in the median cavity.

Bar: I 0 tim

Fig. 14. Close to the wound surface, the ER of a cut sieve cell is swollen (arrow). SC Sieve-cell lumen, w cell wall. x 2,500

Fig, 15. In appropriate electron micrographs the ER appears on either side of a sieve area and consists of tightly convoluted tubules of smooth ER. The small diameter of the sieve pores (small arrows) and the extension of the median cavity (arrowheads) is obvious in this

oblique section, p Plasfid. Bar: 1 gm

Fig. 16. Close to a cut, the densely arranged tubules of the ER (er) at a sieve area change to numerous vesicles (ver). The sieve pores (small arrows) are constricted by callose. Arrowheads, median cavity. Bar: 1 gm

Fig, 17. The ER at sieve areas appears reduced after prolonged fixation in CLSM. SC Sieve cell lumen, w sieve cell wall between sieve areas.

Bar: 10~tm

Fig. 18. In higher magnification, the ER-fluorescence is continuous through sieve pores (arrow) and median cavity (arrowhead). Bar: 10 gm

A. Schulz: Living sieve cells of conifers 159


uoles (Fig. 8). Some spherical organelles, presumably mitochondria (according to size and shape) are distributed over the parietal cytoplasm of the sieve cells. The sieve areas, when sectioned glancingly, do not show specific fluorescence due to DiOC, but may or may not include mitochondria (Fig. 8).

Mature sieve cells

Contrary to immature ones, the mature sieve elements have sieve areas that are characterized by prominent DiOC fluorescence (Fig. 9). This material seems to fill the pit like depressions and makes their location in the thick, secondary sieve-cell wall obvious. Sieve areas connecting two sieve cells show DiOC fluorescence on either side. Except for a parietal layer with mitochondria (Fig. 11 G) and plastids, the lumen of mature sieve cells does not show DiOC fluorescence (Fig. 9). Plastids are recognized by the fluorescence of their envelope (Fig. 12). Gymnosperm sieve cells have specific rela- tions to Strasburger (= albuminous) cells. If a sieve area leads to such a parenchyma cell, it is one-sided, i.e., sieve pores lead to a median cavity in the mid of the common wall where they split into numerous plasmodesmata (Schulz 1990). Staining of mature phloem with DiOC reveals that the specific sieve element/Stras- burger cell-contacts are characterized by DiOC-positive material on the sieve-cell side only (Fig. 9). Whether two- or one-sided, the sieve areas show fluorescence also in the region of the middle lamella where the median cavity is located (Figs. 12 and 13, compare electron micrographs in Figs. 15 and 16).

Three-dimensional CLSM-imaging of mature sieveareas

In addition to the regular view of the XY-plane, the CLSM allows the formation of images perpendicular to this plane (i.e., in the XZ-plane). While regular images are produced by scanning the laser beam in X- and Y-directions, for XZ-images the scanning of the Y-axis is stopped. The resulting line of the X-scan is then moved vertically through the specimen, i.e., the object stage is lowered by 1 gm steps per each X-line. A tangentially oriented block of the bark is then visualized as radial section and the sieve-area pits are accordingly seen in face view (Fig. 11). The analysis of the 3-D structure of the sieve areas is facilitated by a comparison of the XY- (= optical cross section; Fig. 10) and XZ- (= optical glancing section; Fig. 1 t) images. According to this comparison, DiOC-positive material is like a conical disk and fills the elliptic depression of the sieve area. The fluorescence is homogenous

except for a few non-fluorescent, spherical or elongated inclusions (Figs. 10 B and t 1 F) which are similar to sieve-element plastids found often just outside the pit depression (see Fig. 12). This indicates that some plastids are embedded in the ER-complex (cf. Schulz et al. 1989: Fig. 1 I). The series of XZ-images (Fig. 11 A-G) results from a shift of the observed optical plane from one sieve cell into the next one. This shift starts in the upper cell of Fig. 10 (Fig. 11 A and B), runs through the wall (Fig. I 1 C and D) up to the lumen of the neigh- bouring sieve cell (Fig. 11 E-G). The lining of the plasma membrane with DiOC-positive organelles outside the sieve areas is clearly revealed in the XZ-images (Fig. 11 A, B, and E-G).

Wound response at sieve areas

Close to a wound surface, the ER at sieve areas changes its conformation (cf. Figs. 14 with 13, and 16 with 15). This is obvious in living phloem where the ER of the cut sieve cell changes from a homogenous to a vesi- culate appearance (Fig. 14). The same asymmetric response is visible ultrastructurally. A specimen was fixed for electron microscopy and trimmed close the first (preparatory) cut. On the wound-opposed side of the sieve area, the ER complex is disturbed and consists of loosely packed, spherical vesicles (Fig. t 6). In contrast, if, as was routinely done, the first 5 mm of longitudinal phloem sections are trimmed off after fixation in order to avoid wound effects, either side of a sieve area has a network-like ER-complex that consists of densely arranged ER-tubules (Fig. 15). Prolonged fixation results in a further vesiculation of the ER tubules that make up the complexes, so that only remnants remain (Figs. 17 and 18). DiOC-fluorescence, however, is still present in the sieve pore channel and the median cavity (Fig. 18).

Discussion

The present paper gives evidence that the sieve areas, linking mature sieve cells of gymnosperms symplasti- cally, are in vivo covered on either side by ER. This localization is depicted by confocal fluorescence microscopy and a vital ER-dye. The ER-specific DiOC fluorescence appears also in the median cavity, but not in the pore channel of live material (Fig. 13). The channel itself might be too small to give sufficient fluorescence. Staining of fixed material, however, sometimes also gives positive reactions of this channel (Fig. 18). These in vivo observations agree with appropriate EM- fixations, similarly showing endoplasmic reticulum on

A. Schuiz: Living sieve cells of conifers t6t

either side of the sieve-cell/sieve-cell connection and giving evidence that the ER is continuous through the sieve pores and fuses with that of the adjoining sieve cell in the common median cavity. The principle structure of the gymnosperm sieve area and the membranous continuity of plasmalemma and ER were already demonstrated by Kollrnann and Schumacher (1962, 1963) for Metasequoia gl'yptostroboides and were later often confirmed (see, e.g., Neuberger and Evert 1975, Schulz 1990). The location of the ER-complexes raises pro.blems with respect to the M/inch hypothesis on phloem transport since they seem to be important obstacles to any mass flow. In parallel with discussions on the distribution of P-protein in the angiosperm sieve element, it was argued that the membranes of the ER become passively shifted onto one side of the sieve area due to the pressure release during EM fixation, most recently by Warmbrodt and Eschrich (1985). This argument was supported by micrographs of inadequately fixed sieve cells that were published between 1965 and 1974. The present results, however, show that the ER smoothly overlays the sieve area on either side and therefore excludes the argument of an artifactual shift of these membranes. ER occurs then at only one side of a sieve area when it leads to a Strasburger cell (Fig. 9) (see Neuberger and Evert 1975, Sauter et al. 1976). If the phloem is wounded purposefully, the sieve cells do not respond with a shifting of ER material but, on the contrary, with a swelling of the ER-tubules to vesicle- like structures. This is visible both in live preparation and CLSM (Fig. 14) as well as in electron micrographs (Fig. 16) close to the wound surface of sieve cells. This swelling might be interpreted as a mechanism able to rapidly seat translocating sieve cells in case of inter- ruptions and is as such comparable to the rapid plug- ging of angiosperm sieve pores with P-protein in response to wounding (see Evert 1982). Discussions on the mechanism of phloem transport mostly neglect the variability of the sieve-pore size and structure within the species and the plant kingdom. In the inner region of Dioscorea nodal anastomoses sieve pores some close to plasmodesmata (Behnke 1990b), in the gymnosperms they are generally small (less than 0.8 gm, Schulz 1990) and they may exceed 5 ~tm in some angiosperms (LM: Ailanthus 14 tam, Essau and Cheadle t959; EM: Fagus trunk 6.5 gm, Schutz and Behnke 1986). In addition, there is a basic difference between the sieve areas of gymnosperms and the sieve plates of angiosperms (see Behnke 1986). The former should exert much higher resistance to any solute transfer than

the latter, since the plasmatic path through them is much more complex (see Kollmann 1973: Fig. 25). This path is formed by sieve pores, the median cavity in the region of the middle lamella and another set of sieve pores. In contrast, the angiospermous sieve pore is a single penetration of the sieve-plate walt. The variability in the plant kingdom of the symplastic connec- tions between sieve elements excludes the view that sieve elements simply form a pipe allowing the transport of assimilates in their solvent (i.e., water) without considerable resistance along the path. It is hard to believe that some plants, those with wide sieve pores, have a M/inch-type, pressure-driven phloem transport, and others have a different mechanism. The possibility that gymnosperms have a transport mechanism different from the angiosperms was, however, already con- sidered by Weatherley (1975 b). It is interesting to read that M/inch himself investigated the ease of exudation from different trees and noted that Pinaceae did not exude readily when the phloem was treated as in angiosperm trees. Only one Pinus species allowed exudation and this only after several days when the same region of the phloem was cut repeatedly (M/inch 1930: 125). The function of the extended complexes of ER at the sieve areas is totally unknown. It was discussed that the lattice-like, similarly closely packed ER structures, occurring parietal in Dioscorea sieve elements, might form a kind of membrane reservoir (Behnke 1968). Smooth ER cisternae were often described as forming stacks or convoluted aggregates at the plasma membrane of sieve elements (e.g., Thorsch and Esau 1981) and might simply be the remnants of the endomem- brane system after the typical autolysis of the sieve element-protoplast occurring as the last step of its ma- turing. The formation of stacks of ER-cisternae and of fenestrated sheets over the plasmalemma could, however, as well be an important active tool for the function of the sieve element, notably in its ability to sequester calcium (see Sjolund 1990). In contrast to these sheets, which seem to cover nearly 100% of the plasmalemma of Strepthantus sieve-elements (Sjolund and Shih 1983), the ER-complexes in conifers are restricted to the sieve areas. Here, the ER could be involved in callose synthesis. In gymnospenns and angiosperms the synthesis of catlose constricting the sieve pores is a remarkable response to phloem cuts and presumably prevents the leakage of assimilates after an injury (Evert 1982). It requires Ca 2 + ions that could be stored in the ER (see Fink etal. 1981, Kauss t987). Since angiosperms, though well capable of callose formation, do not show

162

comparable ER complexes at their sieve pores (Behnke and Sjolund 1990), it seems unlikely that gymnosperms need such large ER-reservoirs for this function. It re- mains possible that the sole rote of the ER tubules at sieve pores is their ability to swell, so that the sieve pores can be sealed rapidly in response to wounding before caUose synthesis starts. Much more attractive than an involvement in wound response is an active role of the ER complexes for assimilate uptake and/or translocation. In this context an example of a prominent accumulation of smooth ER is quite interesting where obviously a high ratio of sucrose transfer takes place. The plant parasite Cuscuta shows an accumulation of smooth ER in the tip of its "absorbing hypha" (D6rr 1990). After contacting a sieve element of the host, the tip of this hypha develops wall labyrinths like transfer cells and accumulates stacked or otherwise comptexed ER (D6rr 1990). Com- parable to plasmalemma invaginations of transfer cells, the complexation of ER should also increase the surface of the ER considerably. In conifer sieve cells the localization of enzymes showed nucleoside triphosphatase and glycerophosphatase ac- tivities at the ER complexes at the light and electron microscopic levels (Sauter 1976, 1977). An active role of the ER is also supported by its staining with the cationic, lipophilic dye DiOC. This dye is supposed to mark membranes that have a significant membrane potential, with the negative charge inside. This applies to mitochondria (Matzke and Matzke 1986) and was shown to work with ER-mcmbranes of animal cells (Terasaki et al. 1984) and plant cells as well (Quader and Schnepf 1986, Quader 1990). The activity of nucleoside triphosphatases at the ER-complexes, proton gradients across these membranes and, concomitantly, a high membrane surface might together be taken as an indication that assimilate translocation in gymnosperms does not depend solely upon the processes of loading in source leaves and unloading at sinks, but requires also an energy consumptive step within the path between source and sink. The highly ordered system of potential-active ER-cisternae constitutes a non-plasmatic compartment that is continuous through the sieve area. It is conceivable that this compartment is able to regulate the long distance gradient of assimilates in the phloem by re-es- tablishing or steepening the gradient in each sieve cell. This interpretation resembles the relay hypothesis of phloem transport proposing "functional units" in the sieve-tube system of angiosperms (Lang 1979). Trans- port would be discontinuous and energy-consumptive,

A. Schulz: Living sieve cells of conifers

since the units are supposed to be separated by an apoplastic step where solutes must cross two cell membranes and hereby unloaded and reloaded (Lang 1979, Aikman 1980, but see Murphy and Aikman 1989). In conifers, at the sink end of one sieve cell assimilates could be unloaded into the apoplastic ER-compart- merit a n d - after passing the sieve area within the ER- reloaded at the apical end of the next sieve cell. The swelling of the ER close to wounds is indicative for a high osmotic value in this compartment. Pressure release in the cut sieve cell should cause an influx of water into the ER, and thus, its swelling. This system could build up the source/sink gradient in each sieve cell. A longitudinal movement of water across the sieve areas, implied by the M/inch hypothesis, seems not necessary. A lateral influx of water for equilibration of the osmotic and water potentials (see Eschrich and Eschrich 1989) could occur according to the respective sucrose con- centration (see also Lang 1983). This model might be denoted as an intracellular relay model of phloem transport, and would apply to those plants only that are provided with ER-tubules regularly traversing the sieve pores, i.e., to mosses (Scheirer 1990), to a great number of seedless vascular plants (Evert 1990) and to gymnosperms (conifers, Schutz 1990; cycads and genetophytes, Behnke 1990 a). An open question of the present proposal is whether and how the symplastic route of the sieve pore, i.e., the space between sieve-pore plasmalemma and ER, participates in the proposed mechanism. Plasmodesmata and sieve pores interconnect nearly all cells of a higher plant and, like gates, allow commu- nication and solute transfer in the so-formed symplasm. The sieve pores might be regarded as the specialization of plasmodesmata for long-distance transport. Ac- cordingly, their differentiation starts in the plasmodesmata of nucleate phloem cells. In gymnosperms, ER-tubules-presumably arising from the desmotu- bules-still traverse the sieve pores at maturity and further narrow down the rather small pore lumina. In angiosperms, a new quality of symplasmic contact is realized between sieve-tube members. Their sieve pores are generally wider than that of gymnosperms and, moreover, do not obligatorily (but only occasionally) enclose ER-membranes. This is not a function of the pore width, since the smallest known sieve pores in angiosperms, those between nodal anastomeoses of Dioseorea, appear free of ER at maturity (see Behnke 1990b: Figs. 24 and 25). It might therefore be con- cluded that the disintegration of the ER-tubule during the differentiation of a sieve pore out of a plasmodesma

A. Schulz: Living sieve cells of conifers 163

is a new character of angiosperms. This achievement in evolution means a higher degree of plasmatic contact between sieve elements and, at the same time, a decrease in cellular independence.

Acknowledgements Discussions with Prof. Dr. R. Kollmann, Kiel, and financial support from the Deutsche Forschungsgemeinschaft are gratefully acknowl- edged.

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