Planta 9 Springer-Verlag 1982

Planta (1982) 155:377-387

Studies on the leaf of Amaranthus retroflexus (Amaranthaceae): ultrastructure, plasmodesmatal frequency, and solute concentration in relation to phloem loading David G. Fisher* and Ray F. Evert** Department of Botany, Universityof Wisconsin,Madison,WI 53706, USA

Abstract. Both the mesophyll and bundle-sheath cells associated with the minor veins in the leaf of A m a r a n thus retroflexus L. contain abundant tubular endoplasmic reticulum, which is continuous between the two cell types via numerous plasmodesmata in their common walls. In bundle-sheath cells, the tubular endoplasmic reticulum forms an extensive network that permeates the cytoplasm, and is closely associated, if not continuous, with the delimiting membranes of the chloroplasts, mitochondria, and microbodies. Both the number and frequency of plasmodesmata between various cell types decrease markedly from the bundle-sheath - vascular-parenchyma cell interface to the sieve-tube member - companion-cell interface. For plants taken directly from lighted growth chambers, a stronger mannitol solution (1.4 M) was required to plasmolyze the companion cells and sieve-tube members than that (0.6 M) necessary to plasmolyze the mesophyll, bundle-sheath, and vascular-parenchyma cells. Placing plants in the dark for 48 h reduced the solute concentration in all cell types. Judging from the frequency of plasmodesmata between the various cell types of the vascular bundles, and from the solute concentrations of the various cell types, it appears that assimilates are actively accumulated by the sieve-tube - companion-cell complex from the apoplast. Key words: A m a r a n t h u s Leaf ultrastructure Phloem loading - Plasmodesmata - Veins (minor).

Introduction Despite an increasing interest in recent years in the pathways and mechanisms involved in the movement * Present address: NorthCentralForestExperimentStation, U.S.

Department of Agriculture, Forest Service, Rhinelander, WI 54501, USA ** To whomcorrespondenceshouldbe addressed

of assimilates from photosynthetic cells to the sieve tubes in minor veins of leaves, our understanding in this area of plant research remains woefully limited. Two contrasting pathways have been proposed for assimilate movement from the mesophyll to the sieve tubes: an entirely symplastic pathway (Cataldo 1974; Ziegler 1974), and a pathway along which the assimilates temporarily enter the apoplast before entry into the sieve tubes. Disagreement exists over the site at which sugar manufactured in the mesophyll cells enters the apoplast of sugar beet leaves. Evidence obtained by Kursanov, Brovchenko, and co-workers (Kursanov and Brovchenko 1969; Brovchenko et al. 1976) indicates the surgars formed in the sugar beet mesophyll quickly find their way into the apoplast, whereas that obtained by Geiger et al. (1974) and Giaquinta (1976, 1977) indicates the entry of sugars into the apoplast is probably restricted for the most part to the region near the sieve tubes. Assuming that plasmodesmata are present in sufficient numbers, it is conceivable that photosynthates diffuse from the mesophyll cells to the sieve tubes along concentration gradients within the symplast (Ziegler 1974). According to estimates made by Tyree (1970), plasmodesmata constitute the pathway of least resistance for the diffusion of small molecules between cells. Increasing evidence indicates, however, that assimilates do not follow an entirely symplastic pathway from mesophyll cells to sieve tubes and that they are actively loaded from the apoplast into the sievetube-companion-cell complexes of minor veins (Geiger 1976; Hendrix 1977; Evert et al. 1978; Heyser 1980). Geiger and his co-workers, in particular, have amassed impressive data in support of their view that sucrose is loaded from the apoplast into the sievetube-companion-cell complexes in minor veins of the leaves of sugar beet, a C3 dicotyledon (Geiger 1975, 1976, and literature cited therein). More re-

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D.G. Fisher and R.F. Evert: Phloem loading in the leaf of Amaranthus

cently, Evert et al. (1978) a n d Heyser et al. (1978) have c o n c l u d e d that p h l o e m l o a d i n g in the leaves of maize, a CA m o n o c o t y l e d o n , also involves a n active a c c u m u l a t i o n of sucrose into the s i e v e - t u b e - c o m p a n i o n - c e l l complexes f r o m the apoplast. N o t only was there a higher solute c o n c e n t r a t i o n in the sieve tubes a n d their associated c o m p a n i o n cells (osmotic potential, approx. - 1 8 bar) t h a n in the n e i g h b o r i n g v a s c u l a r - p a r e n c h y m a cells (osmotic potential, approx. - 1 1 bar), b u t the s i e v e - t u b e - c o m p a n i o n - c e l l complexes were virtually isolated symplastically from all other cells of the leaf (Evert et al. 1978). M o r e recently, Heyser (1980) has p r o v i d e d strong, if n o t conclusive, evidence for active l o a d i n g of sucrose f r o m the apoplast in c o r n leaves i n v o l v i n g p r o t o n cotransport. The present study - the f o u r t h in a series dealing with s t r u c t u r e - f u n c t i o n relationships in the leaf of Amaranthus retroflexus L., a C~ d i c o t y l e d o n a n d N A D - m a l i c enzyme species (Fisher a n d Evert 1982a, b, a n d c) - was u n d e r t a k e n to determine (1) the p r o b able pathway(s) of the intermediates or p r o d u c t s of p h o t o s y n t h e s i s between m e s o p h y l l cells, b u n d l e sheath cells, a n d sieve t u b e s ; a n d (2) the sites of active p h l o e m l o a d i n g in the m i n o r veins. T o that e n d two sets of data were sought (1) the n u m b e r a n d frequency of p l a s m o d e s m a t a between cell types o f the m i n o r veins a n d s u r r o u n d i n g tissues; a n d (2) the solute concentrations of the various cell types c o n c e r n e d by plasmolytic methods.

Material and methods

Ultrastructural studies. Leaves of field-grown Amaranthus retroflexus L. plants were prepared for electron microscopy as follows: I-ram-wide leaf strips were fixed for 6 h in either 6b/oglutaraldehyde in 0.05 M sodium-cacodylate buffer, pH 7.0, or a mixture of 6% glutaraldehyde and 2% tannic acid buffered in the same manner, post-fixed overnight in 2% OsO4, dehydrated in ethanol, and embedded in Spurr's Epoxy resin (Spurr 1969). Thin sections were cut on a Porter-Blum MT-2 ultramicrotome (Sorvall Newtown, Conn,, USA), stained with uranyl acetate and lead citrate, and viewed and photographed with a Hitachi (Tokyo, Japan) HU-11C electron microscope.

Studies ofplasmodesmatalfrequency. For studies of plasmodesmatal fi'equency, 20 randomly-selected minor veins and surrounding tissues, fixed and embedded as above, were sectioned in the following manner. Transverse sections 2-btm-thick were mounted on glass slides and stained with 0.05% toluidine blue. For each 2-btm-thick section, several thin sections immediately adjacent to the thick section were mounted on coated slot grids and stained with uranyl acetate and lead citrate. The slides containing the 2-~tm-thick sections were placed in a micro-projector and the images of all eells in each leaf cross-section were traced onto sheets of paper. The numbers of plasmodesmata observed between contiguous cells in the corresponding thin sections with the electron microscope were then recorded directly onto the drawings. Where two or more plasmodesmata shared a single median cavity, each "branch" was counted as a single plasmodesma. In order to be consistent, this

procedure was also followed in the case of the pore-plasmodesmata connections between sieve-tube members and companion cells. Altogether, 13,660 plasmodesmata were counted. Because the drawings were traced to scale, the length of the interface between contiguous cells could be determined. This length was measured with a Dietzgen (Des Plaines, Ill., USA) chartometer for each combination of cells. Thus, the number of plasmodesmata per pm of interface (plasmodesmatal frequency) could be calculated for any combination of contiguous cells, data on plasmodesmatal frequency were collected primarily from 40, transverse, non-serial thin sections, two each of the 20 minor veins. Plasmodesmatal frequency between companion cells and sieve-tube members was determined from both the 40 non-serial transverse sections and 34 serial longitudinal sections through a minor vein.

Plasmolysis studies. The relative solute concentrations of various cell types in minor veins and surrounding tissues were determined by plasmolysis studies. Plasmolysis was considered as that point in an increasing concentration series of mannitol solutions at which 50% or more of the cells of a given type exhibited plasmolysis. A cell was considered to be plasmolyzed if 25% or more of its protoplast surface was separated from the cell wall. The experimental procedures used for the plasmolysis studies have already been described in detail (Fisher and Evert 1982b). Briefly, I-ram-wide strips of leaf tissue from growth chambergrown plants were immersed for 30 rain in mannitol solutions ranging in concentration from 0.3 1.4 M, in 0.1-M steps. The leaf strips were then quick-frozen in an isopentane-methylcyclohexane mixture at -160 ~ C, freeze-substituted for 4 d in methanol at - 78~ C, warmed and fixed in 2% OsO4 in acetone, and embedded in Spurr's epoxy resin (Spurr 1969). Thin sections of the tissue were stained with uranyl acetate and lead citrate, and viewed and photographed with the electron microscope. In general, preservation of freeze-substituted tissues was clearly inferior to that obtained for tissues fixed with glutaraldehyde and OsO4. Some of the leaves used in the plasmolysis studies were obtained from plants ("light" plants) taken directly from lighted growth chambers, while others were taken from plants ("dark" plants) which had been grown in lighted growth chambers, but which had been placed in the dark for 48 h preceeding the experiment. The growth-chamber plants were grown from seeds collected from field-grown plants. Results

Brief description of minor veins and surrounding tissues. As is typical for leaves o f CA plants, the m i n o r veins in the A. retroflexus leaf are each s u r r o u n d e d by a p r o m i n e n t , e h l o r e n c h y m a t i c b u n d l e sheath, which in t u r n is encircled by a layer of r a d i a l l y - a r r a n g e d mesophyll cells. T h e chloroplasts of the b u n d l e - s h e a t h cells are localized next to the i n n e r t a n g e n t i a l wall, while a t h i n layer of c y t o p l a s m lines the outer t a n g e n t i a l wall (Fig. 1). M o s t of the mesophyll cells are in direct c o n t a c t with the b u n d l e - s h e a t h cells (Fisher a n d Evert 1982a). The m i n o r veins are c o m p o s e d of c o m p a n i o n cells, v a s c u l a r - p a r e n c h y m a cells, sieve-tube m e m b e r s , a n d tracheary elements. T h e c o m p a n i o n cells are characterized by very dense c y t o p l a s m a n d a close spatial association with the sieve-tube m e m b e r s (Fig. 1). Vasc u l a r - p a r e n c h y m a cells m a y be distinguished from

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Fig. 2. Transverse section through cell wall between mesophyll cell (above) and bundle-sheath cell (below, showing longitudinal views of plasmodesmata. Unlabeled arrows point to endoplasmic reticulum. V, Vacuole. x 36,610; bar=0.25 ~tm companion cells by their somewhat less dense cytoplasm, relatively large vacuoles, and often a lack of spatial association with the sieve-tube members. In comparison with the vascular-parenchyma and companion cells, the sieve-tube members are quite small and often clear in appearance. Sieve-tube members rarely border bundle-sheath cells, and hence, they occupy a more or less median position within the vein (Fig. 1). Mesophyll and bundle-sheath cells. The highly vacuolate mesophyll cells contain numerous chloroplasts with prominent peripheral reticulum. Chloroplasts of the spongy parenchyma cells have well-developed grana, while those of the palisade parenchyma contain poorly developed ones (Fisher and Evert 1982c). The cytoplasm of the mesophyll cells is always quite dense in appearance and, consequently, m e m b r a n o u s components other than those delimiting chloroplasts, mitochondria, and microbodies are usually difficult to discern. Some dictyosomes, small vesicles, and apparently abundant tubular endoplasmic reticulum (ER) are present. The walls between the mesophyll and bundlesheath cells contain large aggregates of plasmodesmata. Two or more plasmodesmata commonly share a single median cavity, and their desmotubules are in contact (and apparently continuous) with tubular ER on both sides of the wall (Figs. ~ 4 ) . In transverse views of plasmodesmata, the desmotubules are fairly sharply defined at all levels of the wall except near the plasmodesmatal orifices, where the desmotubule and the cytoplasmic annulus stain almost equally

Fig. 1. Transverse section of portion of minor vein and surrounding tissues. BS, Bundle-sheath ceils; CC, companion cell; CP, chloroplast; IS, intercellular space; MS, mesophyll cell; S, sieve-tube member; V, vacuole; VP, vascular-parenchyma cell; T, tracheary element, x 2,690 ; bar = 3.7 pm

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D.G. Fisher and R.F. Evert: Phloem loading in the leaf of Amaranthus

Figs. 3, 4. Portions of mesophyll cell and bundle-sheath cell and part of the wall between them, showing transverse views of plasmodesmata and network of tubular endoplasmic reticulum in bundle-sheath cell. BS, Bundle-sheath cell; CW, cell wall; D, desmotubule; MCV, median cavity; MS, mesophyll cell; MT, microtubule; MVB, multivesicular body. Unlabeled arrows point to tubular endoplasmic reticulum. Fig. 3, x 50,000; bar=0.20 pm. Fig. 4, x 94,860; bar=0.10 gm dense (Figs. 3, 4). The p l a s m o d e s m a t a do not appear to have neck constrictions. In bundle-sheath cells, the mostly smooth, tubular E R forms a complex n e t w o r k extending f r o m the

parietal cytoplasm bordering the outer tangential wall (Figs. 3, 4), along the n a r r o w cytoplasmic layer lining the radial walls (Fig. 1), and throughly permeating the organelle-rich region bordering the inner tangen-

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Figs. 5, 6. Portions of bundle-sheath cells. CH, Chloroplast; MC, mitochondrion; PR, peripheral reticulum Fig. 5. Shows extensive network of tubular endoplasmic reticulum (arrows) permeating organe[le-rich cytoplasm, x44,440; bar=0.22 gm Fig. 6. Shows portion of chloroplast with apparent protuberances of outer delimiting membrane (arrows). x 65,830; bar-0.15 gm tial wall (Fig. 5). The tubular E R is often closely associated spatially with the cell organelles. M o r e over, quite frequently small, tubular protuberances appear to extend o u t w a r d f r o m the outer delimiting

m e m b r a n e o f the chloroplast envelope, especially in regions next to peripheral reticulum (Figs. 6, 8), and to come into contact with the ER. W h e t h e r such protuberances are continuous with or merely closely

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D.G. Fisher and R.F. Evert: Phloem loading in the leaf of Amaranthus

Figs. 7-10. Portions of bundle-sheath ceils (Figs. 7, 8) and cells of minor vein (Figs. 9, 10). CH, Chloroplast; CW, cell wall; ER, endoplasmic reticulum; MC, mitochondrion; MB, microbody; P, P-protein; PR, peripheral reticulnm; V, vacuole Fig. 7. Shows apparent tubular protuberances of delimiting membranes of microbody (arrowheads) and mitochondria (arrows'). x 45,290 ; bar =0.22 gm Fig. 8. Shows apparent tubular extensions of mitochondrial cristae (arrows) and protuberances emanating from chloroplast (arrowheads). x 33,680; bar=0.30 gm Fig. 9. Longitudinal views of plasmodesmata in wall between bundle-sheath cell (above) and vascular-parenchyma cell (below). x 43,800; bar =0,23 gm Fig. 10. Longitudinal views of sieve-tube (above) - companion-cell (below) connections. • 24,320; bar = 41 gm

D.G. Fisher and R.F. Evert: Phloemloadingin the leaf of Amaranthus appressed to the tubular ER could not be determined. 1-he outer membrane of the chloroplast envelope is, however, conspicuously thicker and commonly stains more intensely than the tubular ER. The mitochondria and microbodies of the bundlesheath cells also exhibit numerous tubular protuberances, which trail away from the delimiting membranes of these organelles (Fig. 7). As with the delimiting membranes of the chloroplasts, those of the mitochondria and microbodies stain more intensely than the tubular ER. The mitochondria also commonly exhibit protuberances that appear to be extensions of the cristae (Fig. 8). This type of protuberance stains much more intensely than either the delimiting membranes of the mitochondria or the tubular ER. In some instances, the cristae protuberances appear more-or-less short and bulbous; in others, more attenuated. Whether the protuberances emanating from the microbodies and mitochondria are continuous with the tubular ER is an unanswered question. It is clear, however, that the two types of tubules often are in close spatial association with one another. The tubular ER of the bundle-sheath cells apparently is continuous with the desmotubules of plasmodesmata leading to the vascular-parenchyma cells. These plasmodesmata (Fig. 9) are similar in appearance, though not so numerous, as those between the mesophyll and bundle-sheath cells. Like the plasmodesmata between mesophyll and bundle-sheath cells, they occur in aggregates.

Cells of minor veins. The vascular-parenchyma cells have very dense cytoplasm and, hence, as in the case of the mesophyll cells, it is difficult, or sometimes impossible, to distinguish ER and other membranous components within them. As mentioned previously, the vascular-parenchyma cells are highly vacuolated. In addition, they contain a fair number of mitochondria and chloroplasts with well-developed grana but no peripheral membranes (Fisher and Evert 1982c). Plasmodesmata traversing the vascular-parenchyma companion-cell wall are similar in appearance and distribution to those in the walls between mesophyll and bundle-sheath cells. This is also true of the plasmodesmata between vascular-parenchyma cells and between companion cells. The companion cells commonly have denser, less vacuolate protoplasts than the vascular-parenchyma cells, and contain many more mitochondria. In addition, their chloroplasts differ from those of the vascular parenchyma cells in their possession of peripheral lamellae (Fisher and Evert 1982c). Mature sieve-tube members are lined with a parietal layer of cytoplasm containing a network of ER, plastids (Fisher and Evert 1982c), and mitochondria. -

383

Variable amounts of P-protein are also present. The protoplasts of sieve-tube members are interconnected by simple sieve plates on transverse to slightly oblique end walls. Connections between sieve-tube members and companion cells are typical of those between such cell types in other species (Esau 1977), consisting of a pore on the sieve-tube side and one or more plasmodesmata (usually branched) on the side of the companion cell (Fig. 10). Similar connections occur but rarely between sieve-tube members and vascular parenchylna cells. The connections between sieve-tube members and parenchymatic elements, including companion cells, appear to be more or less randomly distributed.

Frequency of plasmodesmata between cell types. A summary of the number and frequency of plasmodesmata between cell types of the minor veins and surrounding tissues is given in Table 1. Both the greatest number and frequency of plasmodesmata occur between mesophyll cells (MS) and bundle-sheath cells (BS), with the next greatest frequency between different cell types occurring between the bundle-sheath cells and vascular-parenchyma cells (VP). Within the minor vein, the greatest fi'equency of plasmodesmata occurs between vascular-parenchyma cells, followed by that of plasmodesmata between vascular-parenchyma cells and companion cells (CC), and then by the pore-plasmodesmata connections between sieve-tube members (S) and companion cells. Each succeeding frequency is only about half that for the previous cell combination. The numbers of plasmodesmata per unit interface (plasmodesmatal frequencies) between four pairs of cell types (MS-BS, BS-VP, VP-CC, and CC-S) were compared with an analysis of variance (Sokal and Rohlf 1969) and were found to be highly significantly different (F3,36 = 150.2). Pair-wise comparisons (MS-BS versus BS-VP, BS-VP versus VP-CC, and VP-CC versus CC-S) using Mann-Whitney U tests and Student's t tests (Sokal and Rohlf 1969) showed all the compared plasmodesmatal frequencies to be highly significantly different (P< 0.0044 in all three comparisons with either test). Identical tests for the same interfaces were conducted for the average number of plasmodesmata per vein per section, resulting in even greater significant differences in all cases. Numbers and frequencies of plasmodesmata between cells of other combinations in the vein are inconsequential. In order to check on the relatively low frequency of connections between companion cells and sievetube members, serial longitudinal sections of an entire sieve-tube member and its associated companion cells were examined. A total of 496 connections were counted between the two cell types. Had serial trans-

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Table 1. Summary of data on number or frequency of plasmodesmata between cell types of minor veins and surrounding tissues in the leaf of Amaranthus retroflexus. For each combination of cells, the upper figure is the average number of plasmodesmata per vein per section, and the lower figure is the frequency, or number of plasmodesmata per gm of interface per section

Mesophyll cells Bundle-sheath cells

Bundle-sheath cells

Vascular-parenchyma cells

Companion cells

Sieve-tube members

249.99+_71.64" 1.09_ 0.26

_b

b

b

40.75 _+17.94 0.18_+ 0.48

Vascular parenchyma cells

23.30 -+ 12.84 0.53--+ 0.31

2.10 _+2.45 0.11_+0.17

5,00 + 7.10 0.59_+ 0.66

12.55 -+8.37 0.25_+0.20

0.65_+0.93 0.05 _+0.09

1.35 _+2.01 0.02 _+0.04

5.10_+5.23 0.13_+0.11

Companion cells

b

a Standard deviation. The relatively large standard deviations for plasmodesmatal frequencies are caused by the fact that in most cases plasmodesmata occur in aggregates, so that a given section may, at the extremes, intercept the center of an aggregate or miss it entirely. Similarly, the considerable size of the standard deviations for the average numbers of plasmodesmata per minor vein per section is caused not only by plasmodesmatal distribution but also by vein size: in the twenty veins sampled, the total number of non-tracheary vascular cells (vascular-parenchyma cells, companion cells, and sieve-tube members) varied from 9 to 33 per vein b Cell association absent or rare

Table 2. Lowest molar mannitol solutions inducing plasmolysis of 50% or more of different cell types in the leaf of "light" and "dark" plants of Amaranthusretroflexus, and relative numbers of starch grains in plastids of those cell types Cell types

Mesophyll cells Bundle-sheath cells Vascular parenchyma cells Companion cells Sieve-tube members

"Light" plants

"Dark" plants

Mannitol (M)

Starch grains

Mannitol (M)

Starch grains

0.6 0.6 0.6 1.4 1.4

abundant abundant abundant abundant a

0.5 0.5 0.3 0.8 0.8

rare common rare rare

a Does not apply

verse sections been m a d e o f t h e sieve-tube m e m b e r , which was 57 ~tm long, one w o u l d have e n c o u n t e r e d a b o u t 0.78 c o n n e c t i o n s p e r sieve-tube m e m b e r p e r section. This is a l m o s t the same as the a v e r a g e value (0.80 c o n n e c t i o n s p e r sieve-tube m e m b e r p e r section) o b t a i n e d for the s i e v e - t u b e - c o m p a n i o n - c e l l c o m b i n a t i o n s o f the non-serial, t r a n s v e r s e sections.

Plasmolysis studies. T a b l e 2 s u m m a r i z e s the results o f the p l a s m o l y s i s studies. F r o m the l e f t - h a n d c o l u m n , it can be seen that, in o r d e r to p l a s m o l y z e t h e c o m p a n i o n cells a n d sieve-tube m e m b e r s o f p l a n t s t a k e n directly f r o m lighted g r o w t h c h a m b e r s , a s t r o n g e r m a n n i t o l s o l u t i o n (1.4 M) was r e q u i r e d t h a n was necessary to p l a s m o l y z e v a s c u l a r - p a r e n c h y m a cells, b u n d l e - s h e a t h cells, o r m e s o p h y l l cells a s s o c i a t e d with the same bundles. The m e s o p h y l l , b u n d l e - s h e a t h , vascular-parenchyma, and companion-cell chloroplasts o f all " l i g h t " p l a n t s c o n t a i n e d n u m e r o u s starch grains (Table 2).

Results o f t h e p l a s m o l y s i s studies on p l a n t s k e p t in the d a r k for 48 h p r i o r to i n i t i a t i o n o f the experim e n t s a r e s u m m a r i z e d in the r i g h t - h a n d c o l u m n o f T a b l e 2. W i t h the " d a r k " plants, c o m p a n i o n cells a n d sieve-tube m e m b e r s were p l a s m o l y z e d b y a 0.8 M m a n n i t o l solution, v a s c u l a r - p a r e n c h y m a cells b y a 0.3 M solution, a n d b u n d l e - s h e a t h a n d m e s o p h y l l cells b y a 0.5 M solution. Thus, all cell types e x a m i n e d in the " d a r k " plants p l a s m o l y z e d at lower m a n n i t o l c o n c e n t r a t i o n s t h a n c o r r e s p o n d i n g cell types in the " l i g h t " plants. In a d d i t i o n , starch reserves in the " d a r k " p l a n t s were largely d e p l e t e d in m e s o p h y l l , v a s c u l a r - p a r e n c h y m a , a n d c o m p a n i o n - c e l l chloroplasts, while t h o s e o f b u n d l e - s h e a t h cells a p p e a r e d to be o n l y slightly decreased, if at all.

Discussion

Mesophyll and bundle sheath. A l t h o u g h it is difficult to discern in the dense c y t o p l a s m o f the m e s o p h y l l

D.G. Fisherand R.F. Evert: Phloemloadingin the leaf of Amaranthus cells, tubular endoplasmic reticulum (ER) apparently is abundant in both mesophyll and bundle-sheath cells of the A. retroflexus leaf, and is continuous from mesophyll cell to bundle-sheath cell via the abundant plasmodesmata in their common wall. As is typical of NAD-malic enzyme species (Chapman et al. 1975; Hatch et al. 1975), the organelles of the bundle-sheath cells exist in close spatial association and have a marked centripetal distribution relative to the vascular tissues. Not reported previously is the extensiveness of the network of tubular ER, which permeates the cytoplasm of the bundle-sheath cells in A. retroflexus and comes in at least close contact with the chloroplasts and mitochondria. By contrast, in an earlier study, Karpilov and Bil' (1976) found virtually no ER in the bundle-sheath cells of A. retroflexus. Unlike the bundle-sheath mitochondria of other groups of C4 plants, those of NAD-malic enzyme type species such as A. retroflexus play a major role in photosynthetic carbon assimilation (Hatch and Kagawa 1974; Rathnam and Chollet 1980). As noted by Hatch and Kagawa (1974), this role demands large fluxes of various carbon compounds into (aspartate) and from (pyruvate and CO2) the mitochondria. Hatch and Kagawa suggested that the large number and size of the mitochondria and the high degree of convolution of their inner membranes may be necessary to provide sufficient surface area for this traffic. Perhaps the close spatial association, if not direct continuity, of the tubular ER with the outer mitochondrial membrane, or even the numerous cristae protuberances observed during the present study, facilitate the movement of substances to and from the mitochondria. Although very common in the bundle-sheath cells, cristae protuberances were not encountered in any other cell type of the A. retroflexus leaf. Evidence was found for the presence of direct connections between chloroplasts and desmotubules via the ER in both mesophyll and bundle-sheath cells of the maize leaf (Evert et al. 1977). Being an NADPmalic enzyme type species, such connections in maize might provide a direct pathway for photosynthetic intermediates to move from mesophyll chloroplast to sheath chloroplast, or vice versa, via the intracisternal space of the ER. Similar connections are unlikely to exist between chloroplasts of these two cell layers in NAD-malic enzyme type species. During a study of the possible pathways of intercellular transport of substances between the mesophyll and bundle-sheath cells of the leaves of maize and A. retroflexus, Brovchenko and Zavyalova (1978) found relatively high amounts of aspartate and extremely low quantities of alanine in extracts from the apoplast. From these results, Brovchenko and

385 Zavyalova concluded that aspartate follows an apoplastic pathway from the mesophyll cells to the bundle-sheath cells, while the reverse transport of alanine occurs through the plasmodesmata. Although these different pathways might be feasible for A. retroflexus, whose bundle-sheath cells lack a suberin lamella, apoplastic movement of any substance across the mesophyll - bundle-sheath interface in the maize leaf might be expected to be greatly inhibited by the suberin lamella in the outer tangential and radial walls of its bundle-sheath cells (Evert et al. 1977).

Structure and frequency of plasmodesmata. With the exception of the plasmodesmata-pore connections between parenchymatic elements and sieve tubes, the plasmodesmatal connections between various cell types in the leaf of A. retroflexus are very similar in structure. Unlike the plasmodesmata between mesophyll cells and bundle-sheath cells of maize (Evert et al. 1977) and Salsola kali (Olesen 1979), those in A. retroflexus apparently lack sphincters, or sphincter-like structures, and neck constrictions. Near the plasmodesmatal orifices, however, the desmotubules and cytoplasmic annuli stain equally dense, obscuring the structure of the ptasmodesmata in this region, the likely sites of sphincters, should any exist. It is the great frequency of plasmodesmata in the mesophyll - bundle-sheath cell walls of C4 plants that has led to their implication in the intercellular transport of the intermediates of photosynthesis between the two layers of chlorenchymatous cells (Osmond and Smith I976). In A. retroflexus, aspartate might be loaded into the ER of the mesophyll cells and then transported to the mitochondria of the bundle-sheath cells via the desmotubules and intercellularly connected ER. If both cytoplasmic annulus and desmotubule serve as transport channels across the wall, one could visualize an opposite flow, say, of alanine, from the bundle-sheath cell to the mesophyll cell, via the cytoplasmic annulus. Inasmuch as the bundle-sheath cell walls of A. retroflexus and other C4 dicotyledons are not suberized (Crookston 1980), apoplastic movement of substances between mesophyll and bundle-sheath cells in this group of C4 plants cannot be precluded. Beginning with the bundle-sheath - vascular-parenchyma interface, both the average number and frequency ofplasmodesmata undergo a significant decrease with increasing proximity to the sieve-tube members. The relative number of connections between companion cells and sieve-tube members is still considerably greater than that between vascular-parenchyma cells and sieve-tube members, as is often the case for minor veins of leaves (Gunning 1976). Nevertheless, the frequency of connections between

386 c o m p a n i o n cells and sieve-tube members in A. retroflexus apparently is quite low c o m p a r e d to that between similar cell types in Beta vutgaris (Geiger et al. 1973), Viciafaba and Tussilagofarfara ( G u n n i n g et al. 1974) and Zea mays (Evert et al. 1978). A l t h o u g h some assimilates could m o v e entirely symplastically f r o m the bundle-sheath cells to the sieve tubes via either or b o t h the v a s c u l a r - p a r e n c h y m a cells and comp a n i o n cells in A. retroflexus, the relatively low n u m b e r s of connections between the pertinent cell types m a k e it, in our opinion, an unlikely m a j o r pathway.

Plasmolysis studies. The results o f the plasmolysis studies support the view that sugar is actively loaded f r o m the apoplast into the sieve tube - companion-cell complexes of the m i n o r veins of leaves (Geiger 1975, 1976; Evert et al. 1978). The bundle-sheath cells and vascular-parenchyma cells of the " l i g h t " plants plasmolyzed in a considerably weaker mannitol solution (0.6 M) than their contiguous sieve-tube m e m b e r s or c o m p a n i o n cells (1.4 M). Geiger et al. (1973) also used graded mannitol solutions to determine the solute c o n c e n t r a t i o n s o f various cell types in the sugarbeet leaf. In that study, the mesophyll cells bordering the vein and vascularp a r e n c h y m a cells o f the vein plasmolyzed at 0.5 and 0.3 M mannitol, respectively, while the c o m p a n i o n cells and sieve-tube m e m b e r s plasmolyzed at 1.0-1.1 M. Hence, the difference in solute concentration between the sieve-tube - companion-cell c o m plexes and the vascular-parenchyma cells o f sugar beet and the " l i g h t " plants o f A. retroflexus are quite comparable. Placing the A. retroflexus plants in the dark for 48 h resulted in a reduction o f the solute concentration in all cell types. Nevertheless, the concentration of solute in the sieve-tube - companion-cell complexes continued to exceed those o f the other cell types, a l t h o u g h less so than in the case o f the " l i g h t " plants. The lower solute concentrations in the " d a r k " plants were a c c o m p a n i e d by reduction in the starch content of vascular-parenchyma, c o m p a n i o n , and mesophyll cells in the leaves examined. There was little a p p a r e n t difference in the starch content of bundlesheath cells b e t w e e n " light" and " d a r k " plants, however, indicating that even after 48 h in the dark, considerable c a r b o h y d r a t e reserves were still available for mobilization and transport. In Z e a mays, all starch disappeared f r o m the leaves examined after 48 h o f darkness, and at that time the solute concentrations o f the v a s c u l a r - p a r e n c h y m a cells, c o m p a n i o n cells, and sieve-tube members were the same (Evert et al. 1978).

D.G. Fisher and R.F. Evert: Phloem loading in the leaf of Amaranthus This research was supported by National Science Foundation grants PCM-8003855 and PCM78-03872. We are grateful to Dr. Robert R. Kowal and Dr. Hyun Kang for their assistance with statistical treatment of the plasmodesmatal studies, and to Susan E. Eichhorn for her assistance in preparation of the manuscript.

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Received 20 November 1981 ; accepted 12 April 1982