Planta 137, 215

Planta

224 (1977)

9 by Springer-Verlag 1977

Protein-accumulating Cells and Dilated Cisternae of the Endoplasmic Reticulum in Three Glucosinolate-containing Genera:

Armoracia, Capparis, Drypetes Lise Bolt Jorgensen, H.-Dietmar Behnke, and Tom J. Mabry* Institute of Plant Anatomy and Cytology, University of Copenhagen, Solvgade 83, DK-1307 Kobenhavn K, Denmark and Lehrstuhl fiir Zellenlehre der UniversitS.t, Im Neuenheimer Feld 230, D-6900 Heidelberg, Federal Republic of Germany

Abstract. Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former "myrosin cells") occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed. Key words: Armoracia -- Capparis -- Endoplasmic reticulum Euphorbia Glucosinolates -- Idioblasts - Myrosinase -- Protein-accumulating cells.

Abbreviations." ABB = aniline blue black ; D C - dilated cisternae ;

E M - e l e c t r o n microscopy; E R - e n d o p l a s m i c reticnlum; G M A = glycolmethacrylate ; LM = light microscopy ; MBB = mercuric bromphenol blue; P A C : protein-accumulating cells; P A S - p e r i o d i c acid-Schiff * Recipient of an Alexander von Humboldt Award and in residence at the University of Heidelberg during the period when this research was carried out. Permanent address." Department of Botany and Cell Research Institute, University of Texas, Austin, Texas 78712, USA

Introduction

Specialized protein-storing cells are reported for many taxa within the Capparales (Dahlgren, 1975), an order which is characterized by the presence of glucosinolates (see Ettlinger and Kj~er, 1968). These specialized cells (as revealed, for example, by a strong reaction to Millon's reagent) occur as idioblasts and are most often called myrosin cells (Guignard first introduced this expression in 1890) reflecting the view that they contain myrosinase, the fi-thio-glucosidase which catalyzes the hydrolysis of glucosinolates. However, in a review of Guignard's and others' findings, Ettlinger (unpublished manuscript) considered the evidence insufficient to support or deny the localization of the myrosinase in these specialized cells; therefore, we employ the term "protein-accumulating cells" (PAC). In a series of papers on Sinapis alba and other members of the glucosinolate family Brassicaceae, Iversen (1970a, b; 1973) and Pihakaski and Iversen (1976) attempted to correlate the presence of myrosinase with the occurrence of dilated cisternae (DC) of ER, organelles which in our experience do not occur in the protein-accumulating cells. However, whether or not the proteinaceous material detected in these DC is indeed myrosinase is, in our view, not settled. At the present time DC have been detected in different organs and tissues of more than 2:5 species of the Brassicaceae (Bonnett and Newcomb, 1965; Favali and Gerola, 1968; Iversen and Flood, 1969; Iversen, 1970b; Cresti etal., 1974; Havelange and Courtoy, 1974; Gunning and Steer, 1975, p. 189; Hoefert, 1975) but were not found in several other glucosinolate species" Schivereckia podo!ica (Brassicaceae) and four species of Reseda (Resedaceae) (Iversen, 1970b). There are reports of dilated ER containing some structured material in other than glucosinolate plants

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(e.g. Lance-Nougar~de, 1965; Schnepf and Deichgr/~ber, 1972; Esau, 1975) but the nature of these ER inclusions appears to be different from the DC inclusions in Brassicaceae, except for Eschscholzia (Papaveraceae), where Iversen (1970b) found DC which appeared to be identical to those in Brassicaceae. These conflicting reports concerning the occurrence and roles of DC and PAC in glucosinolate taxa prompted us to investigate still other glucosinolate taxa. In this connection we report here our first results which describe a comparative light (LM) and electron microscopic (EM) investigation of three glucosinolate taxa: Armoracia rusticana (Brassicaceae), Capparis cynophallophora (Capparaceae) and Drypetes roxburghii (Euphorbiaceae). The investigation of Drypetes is of special significance since its usual taxonomic treatment within the Euphorbiaceae has been questioned by Ettlinger (unpublished manuscript). The presence of glucosinolates in D. roxburghii was reported by Kj~er and Friis (1962). All three species were examined for both protein-accumulating cells and dilated ER-cisternae. It should be noted that protein-accumulating cells were detected as early as 1886 by Heinricher in Armoracia rusticana and that Solereder (1899, p. 83) lists Capparis cynophaIlophora as a species containing PAC. The most recent investigation of PAC is by Worker and Vaughan (1976) on Sinapis alba.

Material and Methods Young stems of Armoracia rusticana Gaertner, Meyer et Scherbius ( = Cochlearia arrnoracia L.), Capparis cynophallophora L. and Drypetes roxburghii (Wall.) Hurusawa (=Putranjiva roxburghii Wall.) from the Botanical Garden, University of Copenhagen, were investigated. For LM investigations thin longitudinal sections were fixed overnight at 4~ C in 3% glutaraldehyde in 0.1 tool 1 1 Na-cacodylate, pH 7.2, washed in the same buffer and then treated further according to Feder and O'Brien (1968). After embedding in glycolmethacrylate (GMA) semi-thin (3 gin) sections were obtained with glass knives on a standard rotary microtome (Reichert OMS). Staining procedures for GMA sections were for protein: aniline blue black (ABB; Fischer, 1968) and mercuric bromphenol blue (MBB; Mazia et al., 1955); for polysaccharides and other vic-glycol-containing substances: periodic acid-Schiff (PAS; Feder and O'Brien, 1968). Millon's reagent (indicative of tyrosine, Baker, 1956) may be used on GMA sections, but the color reaction obtained is not very intense. Therefore, cryostat sections, 12-16 jam, of 4% formaldehyde-fixed material were also used. The sections were examined in a Reichert "Fluorpan" microscope using normal or, usually for GMA sections, phase-contrast optics. For EM observations, thin (100-300 gm) longitudinal handsections about 6-10 mm long, were prefixed for 3 h at 4~ in a 4% paraformaldehyde-5% glutaraldehyde mixture in Na-cacodylate buffer (0.1 mol 1-1 at pH 7.2), washed in the cacodylate buffer, postfixed at room temperature for l h in 1% OsO4, dissolved in the same buffer and dehydrated through acetone to propylene oxide. Before embedding in an Epon/Araldite mixture the distal

parts of the slices were discarded and only the central parts subdivided and used for polymerisation (cf. Behnke, 1975). Also Spurrmedium (Spurt, 1969) was used for embedding after dehydration in ethanol and propylene oxide. Ultrathin sections were cut with glass knives on Reichert OMU 2 or LKB III ultramicrotomes and double poststained with uranyl acetate and lead citrate. The sections were viewed and photographed with Siemens Elmiskop EM t0t or Jeot JEM-T7.

Observations

1. Armoracia LM-observations of Amoracia rusticana stems show protein-accumulating cells (PAC) occurring sporadically as idioblasts both in pith and cortex. They are normally of about the same size as the surrounding cells but may be up to twice as long in the axial direction. The vacuoles in the PAC are Millon-posirive, stain intensely blue with the protein reagents ABB and MBB and are PAS-positive (Fig. 1). These PAC normally contain one large central vacuole (the cell depicted shows shrinking artifacts) and a cytoplasmic layer containing a nucleus and several small vacuoles reacting like the central vacuole to staining procedures. These smaller vacuoles are globoid, 1-4 gm diameter (Fig. 1). The PAC contain no amyloplasts in contrast to the surrounding normal parenchyma cells. At the ultrastructural level the PAC show a large central vacuole with homogeneous granular content, and peripheral to this smaller vacuoles are found (Fig. 2), Occasionally the smaller vacuoles are in direct contact with the central vacuole. In the cytoplasm the dominant element is tubular or cisternal, granular ER with local agranular dilatations (Fig. 2) enclosing a material comparable to that found in the vacuoles. From the appearance of some of the ultrathin sections (e.g. Fig. 3) the ER is very extensive, even to the point of being a dominant structure of the cytoplasm. Plastids with few thylakoids and devoid of starch and mitochondria are found together with dictyosomes. The normal parenchyma cells in the pith are characterized as follows: highly vacuolated cells, the vacuolar content not reactive to the stains used, with a peripheral cytoplasm containing a nucleus, amyloplasts, mitochondria and conspicuous long, slender inclusions (Figs. 4, 5). These more or less curved inclusions, of dimensions 0.5-1 gm in diameter and extending up to 40 lain in length, react with ABB and MBB, but not with PAS. They occur throughout the pith cells and are found less frequently in cortex cells, including collenchyma below the epidermis, and in vascular parenchyma.

L.B. Jorgensen et al. : Protein-accumulating Cells and Dilated Cisternae

217

Fig. 1-3. Armoracia rusticana. Protein-accumulating cells (PAC) Fig. 1. Semi-thin section, periodic acid-Schiff stained, through PAC fi-om pith. Peripheral cytoplasm with small spherical vacuoles (V) reacting PAS-positively like the material in the central vacuole which appears somewhat shrunken. Neighbouring parenchyma ceils with amyloplasts (A). x 1200. Marker= 10 gm Fig. 2. Cytoplasmic area of PAC from pith showing granular ER with dilated areas. Small vacuoles (V) located near the central vacuole. x 20,000 Fig. 3. Cytoplasmic area of PAC from cortex showing abundant granular ER with dilated areas. Central vacuole at CV. M -mitochondria. x 20,000. Marker = 1 ~na

At the EM-level the inclusions are unit-membrane bound organelles with ribosomes attached to the membrane (Fig. 6), They are supposedly of ERorigin, since a continuation to granular ER is occasionally observed. The inclusions are thus identical with the dilated cisternae found by Iversen (1970b) in other Brassicaceae. The DC have a content composed of 24-28 nm wide rod- or tubular-like structures, oriented parallel in a longitudinal section of the DC (Fig. 6). Yransections of the DC show the tubules oriented in an irregular pattern with a centreto-centre spacing of 50-60 nm.

2. Capparis LM-observations of Capparis cynophallophora stems show protein-accumulating cells (PAC) occurring as idioblasts throughout the cortex, pith and vascular parenchyma. They are usually of about the same size as the surrounding normal parenchyma cells but may

be somewhat longer in the axial direction. They sometimes occur in pairs or triplets with the cells superimposed upon each other in the axial direction. The big vacuoles of the PAC turn brick red with Millon's reagent and exhibit strong blue coloration with ABB and MBB (Fig. 7). The vacuoles also react strongly PAS-positive. A peripheral section (Fig. 8) of the same PAC as in Figure 7 shows near the periphery of the large vacuole similarly stained small globoid vacuoles (diam. 0.5-1 gin) together with a nucleus and many mitochondria. The PAC show at the EM-level (Fig. 9) a large, central vacuole with homogeneous, moderately electron-dense granular material. Besides plastids, mitochondria and dictyosomes the surrounding cytoplasmic layer contains a striking system of cisternal or narrow tubular ER which occasionally shows local dilatations. Also vesicles are found which together with the ER have a moderately electron-dense content, resembling the material in the central vacuole. The vesicles (size range ab. 0.4-1.1 ~un) apparently fuse

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L.B. Jorgensen et al. : Protein-accumulatingCells and Dilated Cisternae

Fig. 4-6. Armoraeia rustieana. Dilated cisternae of endoplasmic reticulum (DC) Fig. 4-5. Semi-thin sections, stained with mercuric bromphenol blue (MBB) from parenchyma cells from pith. Cytoplasm with DC and amyloplasts (A). Both x 1500. Marker= 10 gm Fig. 6. Dilated cisterna (DC) with longitudinally oriented material, x 30,000. Marker= 1 gm

with each other and with the central vacuole (Fig. 9). Thus the vesicles could be considered as small vacuoles and identical to the small vacuoles seen in LM. In the phloem most parenchyma cells are characterized by ovoid to cylindrical inclusions (Fig. 10). They are of dimensions t.5 x 4-6 lain, stain blue with ABB and MBB, but do not react with PAS. The inclusions, often occurring in clusters and sometimes with an axial orientation (Fig. 12), are located in the cytoplasm which lines apparently empty vacuoles. They are not found in stem tissues other than phloem parenchyma. At the EM-level the axial phloem parenchyma cells, which in Figure 11 appear adjacent to a sieve tube, in addition to many other organelles show unitmembrane bound, somewhat extended ovoid inclusions. These inclusions are dilated cisternae of ER, presumably homologous to the DC found in Armoracia. In Capparis also the D C membrane has ribosomes attached and, occasionally, connections to granular

E R (Fig. 13). The DC contain densely packed and longitudinally aligned protein tubules, the length of which is determined by the size of the DC and with a diameter ranging up to about 28 nm (Fig. 14).

3. Drypetes LM-investigations on cryostat sections from stems of Drypetes roxburghii do not reveal Millon-positive idioblasts. However, Millon-positive cells are found among phloem parenchyma cells. The GMA-sections which are better preserved show the presence of two types of cells: (1) the ordinary phloem parenchyma cells (Fig. 15: PPC) characterized by the vacuolar content being of a coarse granular structure of variable quantity, probably of tannin nature, and (2) an unusual type, the protein-accumulating cells (Fig. 15 : PAC), which are very long, axially extended cells with vacuoles having a homogeneous content which is MilIon-positive. The vacuolar content also stains blue

L.B. Jorgenset~ et al. : Protein-accumulating Cells and Dilated Cisternae

Fig, 7-9.

219

Capparis c;vnophctllophora. Protein-accumulating cells (PAC)

Fig. 7-8. Two contiguous semi-thin sections of PAC from cortex, stained with aniline blue black (ABB). Figure 7 shows the heavily stained central vacuole surrounded by a thin cytoplasmic layer. Figure 8 shows small vacuoles (V) stained like the central vacuole, part o f which is seen to the left and right of the nucleus (N). x 1500. M a r k e r = 10 ~m Fig. 9. PAC from cortex showing rather electron-dense material in the central vacuole. In the cytoplasm granular cisternal ER and small vacuoles (V) which fuse mutually or with the central vacuole, x 25,000. Marker = 1 gm

with the proteinaceous stains ABB and MBB and is PAS-positive but the reactivity to these stains including the Millon reagent is not as vigorous as observed with the PAC of Armoracia and Capparis. In some of the PAC in Drypetes parts of the vacuoles exhibit darker-stained, faintly striated areas, suggesting a high concentration of protein, orderly arranged (Fig. 15: H). This material shows a weak birefringence when observed in polarized light. EM-observations on phloem again show the two types of phloem parenchyma cells : the ordinary ones with coarse tannin precipitations in the vacuoles (Fig. 16: PPC) and PAC with vacuoles filled with homogeneous filamentous material (Fig. 16: PAC). The same filamentous content is also frequently encountered in small vacuoles which lie inside the parietal cytoplasm, next to the central vacuole and which are occassionally seen to fuse with the latter (Fig. 18). In the PAC the vacuoles leave space only for a thin layer of cytoplasm along the axial walls (Fig. 17). Here and near the polar parts of the cell where the

cytoplasm is more extensive, are many organelles: nucleus, mitochondria, proplastids, dictyosomes and granular cisternal ER. Also dilated areas of ER are found. Figure 19 shows cisternal granular ER~ tubular parts of granular ER and small vacuoles which cont a i n - i n different a m o u n t s - filamentous material identical to that found in the central vacuole. Indications for a derivation of vacuoles from cisternal ER and a synthesis of filaments by the ER is hereby given: As suggested in Figure 20, the cisterhal ER at one end seems to be blown up like a balloon to give rise to a small vacuole, ready to fuse with the large central one. Both the cisterna and the small vacuole contain identical filaments, in longitudinal orientation in the former, while beginning to be distributed in the latter. Figure 2t gives similar information of the ER-vacuoIe relations. Two cisternae which are sectioned show densely packed, longitudinally oriented filamentous contents (Fig. 21: I, II). In some parts of cisterna No. II the section passes alongside the surface of the ER membrane thus giv-

220

Fig. 10-14.

L.B. Jorgensen et al. : Protein-accumulating Cells and Dilated Cisternae

Capparis cynophallophora. Dilated cisternae of endoplasmic reticulum (DC)

Fig. 10M1. Phloem. Sieve-tube member (ST) and companion cell (CC) together with phloem parenchyma cells (PPC) which contain DC of ovoid to cylindrical shape. Figure 10. Light microscope, aniline blue black (ABB). x 1500. Marker= 10 vtm. Figure 1h Electron microscope. • 5000. Marker = 1 gm Fig. 12. Mercuric bromphenol blue (MBB) stained semi-thin section. Phloem parenchyma cell with longitudinally oriented DC forming a cluster, x 2000. Marker= 10 ~_tm Fig. 13. Phloem parenchyma cell with DC showing connection to normal, granular ER. x 25,000. Marker = 1 gm Fig. 14. DC from phloem parenchyma cell. The enclosed material is composed of tightly packed longitudinally oriented rods or tubules. • 37,000. Marker= I gm

Fig. 15-19.

Drypetes roxburghii. Protein-accumulating cells (PAC)

Fig. 15, Semi-thin section through phloem stained with mercuric bromphenol blue (MBB). Two different types of phloem parenchyma cells are shown, ordinary phloem parenchyma cell (PPC) having vacuoles with coarse granular tannin precipitations and protein-accumulating cell (PAC) in which the vacuoles have a homogeneous content (marked *). The vacuoles in this PAC exhibit darker-stained, faintly striated areas (marked II). x 2000. Marker= 10 gm Fig. 16. Phloem, Sieve-tube member (ST), companion cell (CC) belonging to a sieve-tube member not within the section, ordinary phloem parenchyma cell (PPC) with coarse tannin precipitations in the vacuole, and protein-accumulating cell (PAC) with a homogeneous vacuolar content, x 10,000. Marker= 1 gm Fig. 17. Lateral part of PAC, central vacuole towards right. Cytoplasm with abundant granular ER. The vacuolar material is composed of short filaments which in most of the vacuole exhibit a random orientation (marked *) but towards the tonoplast (T) occur with a longitudinally aligned parallel orientation (marked I1)- • 20,000. Marker= 1 ~m

Fig. 18, Lateral part of PAC showing fusion of tonoplast m e m b r a n e (T) with the membrane of a small elongated vacuole which contains the same filamentous material as the central vacuole. Located in the cytoplasm another small vacuole at V. • 25,000. Marker = 1 g m Fig. 19. PAC, cytoplasmic area below and part of central vacuole above. Cisternal granular ER, small tubular parts of granular ER and small vacuoles (V). The vacuoles contain the filamentous material in different amounts. • 25,000. Marker 1 g m

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Fig. 20 and 21. Drypetes roxburghii. Protein-accumulating cells Fig. 20. PAC, polar cytoplasmic part. Cisternal, straight ER expands to produce a small vacuole. Filamentous material is encountered in both the cisternal and expanded parts.of ER. M=mitochondria. x 40,000. Marker= 1 gm Fig. 21. PAC, polar cytoplasmic part showing cisternae of ER in different planes of cutting. To the left the granular surface of cisternal ER. In the middle a transected cisterna (I) showing tightly packed filamentous material inside the cisterna. Cisterna II is a straight part of ER. Obviously the vacuole (V) below cisterna II has the same relationship to the straight part as shown in Figure 20. x 40,000. Marker = 1 gm ing the appearance of a direct connection between cisterna and vacuole. Figure 21 also demonstrates that the cisternal parts o f the v a c u o l e - E R connection are still studded with ribosomes, albeit in reduced number, as c o m p a r e d with other cisternae (Fig. 21 : ER). The richness of cisternal E R in the plasmatic area o f these cells is in support o f the assumption that these axial p a r e n c h y m a cells are specialized in protein accumulation, synthetized in the cisternal parts o f the E R and secreted into the central vacuole. The L M - h i s t o c h e m i c a l tests imply that the vacuolar content in the P A C is c o m p o s e d of proteins (ABB and M B B tests) and of vic-glycols (PAS test). The observed filaments have a diameter o f a b o u t 10 n m a n d a length o f 350 to 550 n m or more. C o m p a r e d

with other types of filaments (e.g. microfilaments or protein filaments o f sieve elements) these vacuolar filaments in the P A C o f Drypetes are relatively straight and rigid appearing like little needles; they m a y be either r a n d o m l y distributed in a criss-cross pattern or evenly aligned parallel to the cell axis; areas with a strong parallel orientation o f the filaments at the periphery o f the vacuole (Fig. 17: II) m a y a c c o u n t for the weak birefringence observed in the polarizing microscope. Discussion

The aim o f the present investigation was to determine whether or not a m o r e general correlation could be f o u n d between the presence o f glucosinolates, the oc-

L.B. Jorgensen et al. : Protein-accumulatingCells and Dilated Cisternae currence of protein-accumulating cells and the formation of specific dilated cisternae of ER, utilizing as test species Armoracia rusticana, Capparis cynophallophora and Drypetes roxburghii. Our investigations clearly demonstrated the presence of PAC in stem tissue of Armoracia and Capparis. These cells are idioblasts scattered through cortex, pith and vascular parenchyma and contain large central vacuoles which are MBB and PAS positive. As revealed by EM, the PAC have a homogeneous, finely granular content and abundant granular ER in the cytoplasm. In addition, the ER-cisternae which locally swell to produce agranular dilatations contain a material with the same staining properties as that found in the central vacuole. This material is also found in vesicles of different sizes ranging from the size of the local dilatations of ER to that of small vacuoles. The vesicles fuse mutually or with the central vacuole. The transfer of material from ER to the central vacuole is thus apparently mediated by vesicles. Both Armoracia and Capparis also contain DC: in the stem of Armoracia rusticana throughout cortex and pith cells, in the stem of Capparis cynophallophora in phloem parenchyma. By staining with protein-specific reagents the DC in both species were initially detected in LM sections. The semi-thin sectioning technique used here to demonstrate the presence of DC is of great advantage in not only indicating the chemical nature of their contents but also in clearly delineating their longitudinal extension which in EM preparations remains obscure due to the ultrathinsectioning. LM as well as EM pictures disclose a morphological difference between the DC for the two species. While the extended DC of Armoracia rusticana appear similar to the structure of DC in other Brassicaceae, the phloem parenchyma cells of Capparis cynophallophora contain broader and shorter, more or less ovoid DC. This difference, however, is not family specific (Jorgensen, unpublished res.). The DC of both species contain the same electron dense structures: long protein filaments, about 25 nm in diameter, and arranged parallel to the long axis of the DC, typical of reports from other species. However, a close examination of published micrographs showing DC in different tissues of various Brassicaceae and an unpublished observation on additional species suggest that in the root tip cells the DC content is always amorphous. In Drypetes the situation is different. PAC occurring as idioblasts are not found ; instead the combined application of LM and EM methods reveals PAC as integral parts of the axial phloem parenchyma. The PAC of Drypetes have large central vacuoles which after EM-fixation techniques display fine ilia-

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mentous contents, thus differing from the finely granular material inside vacuoles of idioblastic PAC of Armoracia and Capparis. The filaments (about 10 nm diam. and approximately 500 nm long) are of a relatively constant size and, compared to other protein filaments, appear more rigid. The filaments in the central vacuole presumably originate in the cisternal granular ER, which locally swells to give rise to dilatations or small vacuoles which coalesce with the central vacuole. Thus, the mode of transfer of the structurally different vacuolar material from granular ER to the vacuole apparently occurs in the same way in the two types of PAC. These findings support the hypothesis for the origin of the vacuole from local dilatations of cisternal ER (e.g. Mesquita, 1969; see also review by Matile, 1974). The histochemical tests are identical for the three species except for differences in intensity: much weaker for D~Tpetes (both protein and PAS reactions) than for Armoracia and Capparis. The tests suggest that the vacuolar material might be glycoproteins. However, for Drypetes the finding of the easily recognizable needle-like material inside cisternae of ER and the subsequent release of the same material into the vacuoles without passing through dictyosomes argues against this possibility since during synthesis of glycoproteins the glycosyl moieties are thought to be added to the proteins in dictyosomes (e.g. Neutra and Leblond, 1969; Zagury et al., 1970). Ettlinger (unpublished manuscript) has suggested that the PAC might be sites where a high concentration of glucosinolates could occur, with positively charged vacuolar proteins functioning as a counterbalance to the anionic nature of the glucosinolatcs. Thus another explanation for the PAS-positivity of the vacuoles may be the presence of vic-glycols in the glucose-moiety of the glucosinolates. However, lacking a direct histochemical test for glucosinolates, the homology in this respect of the two cell types in the three species remains obscure. Our observations concerning dilated cisternae also require discussion: in Drypetes cells with DC typical of Brassicaceae were not found although some dilated cisternae were detected inside the PAC. These dilated cisternae of Drypetes contain the same relatively short filaments found in the central vacuole and thus act as a dynamic compartment between the ER and the vacuole. Thus the Drypetes dilated cisternae are unlike the DC in members of the Brassicaceae as well as in Capparis. These latter DC occur in various parenchymatic cells and once formed, they are morphologically distinct from other organelles; they are never found within the protein-accumulating cells. This type of D C - t h e organelle-like Brassicaceae D C - t y p e was first described by Bonnett and Newcomb (t965)

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and systematically surveyed by Iversen (1970 b) within Brassicaceae. This type may be confined to Brassicaceae and Capparaceae since Iversen (1970b) could not demonstrate DC in the closely related Resedaceae, and our preliminary investigations on other glucosinolate families like Bataceae, Gyrostemonaceae, and Salvadoraceae have not revealed this type of DC in stem material. It is our view that too few data are available to clarify the relationships if any, of protein-accumulating cells and dilated cisternae to glucosinolates and myrosinase. Therefore, studies will be continued with still other glucosinolate taxa. We are indebted to Prof. Martin G. Ettlinger, H.C. Orsted Institute, Copenhagen University, for permitting us access to his unpublished manuscript and for informative discussions. This work was supported by Deutsche Forschungsgemeinschaft (grant to H.D. Behnke). We thank Miss B. Schmidt and Miss D. Laupp (Heidelberg) and Lis Munk Frederiksen, Lisbeth Thrane Haukrogh, H. Elsted Jensen, Kirsten Pedersen and S.A. Svendsen (Kobenhavn) for technical assistance during this investigation. T.J.M. acknowledges financial support from the National Institutes of Health (Grant HDO-4488)

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Received 17 May," accepted 31 August 1977