ULTRASTRUCTURE, ORGANIZATION AND CYTOCHEMISTRY …For light microscopy, semithin sections of plastic-embedded tissue were staine 1d wit % Toluidinh e Blue 0 in 1 % borax solution

J. Cell Sci. 64, 231-244 (1983) 231Printed in Great Britain © The Company of Biologists Limited 1983

ULTRASTRUCTURE, ORGANIZATION AND

CYTOCHEMISTRY OF CYTOPLASMIC CRYSTALLINE

INCLUSIONS IN ORIGANUM DICTAMNUS L. LEAF

CHLORENCHYMA CELLS

A. M. BOSABALIDIS1'* AND D. PAPADOPOULOS2

Department of Botany, University of Thessaloniki, Thessaloniki, Greece

Department of Physics, University of Thessaloniki, Thessaloniki, Greece

SUMMARY

In the cytoplasm of chlorenchyma cells in primary Origanum dictamnus leaves, rectangularprism-like crystals occur, which, as shown by pepsin digestion, are proteinaceous. In sections theyusually show either a square lattice or striations running parallel to the short or long axis. In bothcases the interepacings are estimated to be about 100 A, suggesting that the arrangement of thecrystallographic planes possesses tetragonal symmetry. High magnifications of the crystalline in-clusions together with analysis of their diffraction patterns showed that the striations are composedof double helices of protein macromolecules. In leaves 3—4 mm long, eroded crystals are oftenobserved. When leaves are larger than 5 mm, no crystalline bodies can be identified in chlorenchymacells. It is possible that they represent a storage form of protein, which is used later by the developingcells.

INTRODUCTION

Crystalline inclusions of proteinaceous nature have often been observed in plantcells under the light and electron microscopes (Nageli, 1862; Heinricher, 1891; Frey-Wyssling, 1955; Sitte, 1958). These structures are either located within cell organ-elles, such as plastids (Behnke, 1973; Sprey, 1977), mitochondria (Tsekos, 1972;Gailhofer & Thaler, 1975), endoplasmic reticulum (Brunt & Stace-Smith, 1978),microbodies (Frederick, Gruber & Newcomb, 1975; Silverberg, Fischer & Sawa,1976) and nucleus (Schnepf, 1971; Heinrich, 1972), or free in the cytoplasm(Williams & Kermicle, 1974; May & Kiinkel, 1975). Cytoplasmic crystalline in-clusions vary in size and shape. Their outline is usually square, rectangular, rhomboidor polyhedral (Jensen & Bowen, 1970; Burgess, 1971; Metitiri & Zachariah, 1972),while their internal structure usually appears as a square lattice or a honeycomb, orin the form of parallel striations (Schnepf & Deichgraber, 1969; Thaler & Amel-unxen, 1975; Renaudin & Cheguillaume, 1977). In sieve elements of Papilionaceae,P-protein crystals seem to consist of a spindle-like central body, which may or maynot have a long thin tail at each end (Palevitz & Newcomb, 1971; Lawton, 1978;Arsanto, 1982).

•Author for correspondence.

232 A. M. Bosabalidis and D. Papadopoulos

Cytoplasmic crystals present in chlorenchyma cells of Origanum dictamnusmesophyll have been studied in the present investigation by means of electron micro-scopy and cytochemistry in order to determine their form, organization and chemicalconstitution.

MATERIALS AND METHODS

Primary leaves of 12-day-old seedlings of Origanum dictamnus L. (Lamiaceae) were used. Smallpieces of the tissue were fixed in a cacodylate-buffered glutaraldehyde/paraformaldehyde mixture(Karnovsky, 1965) for 3 h at room temperature. After washing in buffer the material was post-fixedin 1 % osmium tetroxide, similarly buffered for 3 h in a refrigerator. Dehydration was carried outin a graded ethanol series and was followed by infiltration and embedding in Spurr's Epoxy resin.For light microscopy, semithin sections of plastic-embedded tissue were stained with 1 % ToluidineBlue 0 in 1 % borax solution. Ultrathin sections for electron microscopy were collected on copperor gold grids covered with a Formvar supporting film. They were stained with uranyl acetate andlead citrate, and examined in a JEOL 100CX electron microscope equipped with a goniometricstage.

For enzyme digestion, ultrathin sections of double-fixed material were treated for 20min with10% H2O2 before incubation in 0-5 % pepsin (Serva, research grade) in CM M-HCI for 3-24 h at37°C. After washing in distilled water the sections were stained as described above (Behnke, 1975).

OBSERVATIONS

Mesophyll chlorenchyma cells of primary leaves are characterized by the presenceof large and numerous chloroplasts usually located in the parietal cytoplasm. Thechloroplast matrix is electron-dense, containing a well-developed grana-free systemwith either a few small starch grains or none at all. The numbers of mitochondria andmicrobodies are normal and close associations between them and the chloroplasts haveoften been observed. The cytoplasm is densely filled with ribosomes. There is a largenucleus. Rough endoplasmic reticular cisternae are short and few in number, whilethe dictyosomes are scarce, producing smooth vesicles with electron-translucent con-tents.

During this stage of leaf development, crystalline inclusions located in theperipheral cytoplasm, or in plasma strands traversing the central vacuole, arefrequently observed in the chlorenchyma cells (Fig. 1). They occur isolated or ingroups and display an organized internal structure. In Fig. 2 an almost square crystalis in contact with two connected elongated ones, which appear broken in their middle

Abbreviations used in figures: cp, chloroplast; cr, crystal; cw, cell wall; d, dictyosome;mb, microbody; nice, mesophyll chlorenchyma cell; n, nucleus; v, vacuole.

Fig. 1. Young mesophyll chlorenchyma cells containing crystalline inclusions (arrows) inparietal cytoplasm or in plasma strands traversing the central vacuole. X6000.

Fig. 2. A nearly square crystal in contact with two elongated ones. The latter probablyunderwent breakage in their middle region, when the square crystal was pushed towardsthem by the central vacuole. The arrow indicates the direction of movement. X 39 000.

Fig. 3. A long rectangular crystal laterally rammed into by another smaller one. Thedirection of ramming is indicated by the arrow. X46 000.

Cytoplasmic inclusions in O. dictamnus leaf cells 233


region. It is possible that the square crystal was pushed by the central vacuole (extend-ing movement) into the rectangular crystal causing it to break. A similar rammingmovement that was probably caused by a chloroplast is shown in Fig. 3.

The crystal contours most frequently encountered are rectangular (Figs 4, 5),trapezoidal (Fig. 6) and triangular (Fig. 7). These different outlines correspond to

cr

Figs 4—7. Crystal contours usually encountered in the cytoplasm of leaf chlorenchymacells. They may be rectangular (Fig. 4, X37 000; Fig. 5, X33 000), trapezoidal (Fig. 6,X34000) or triangular (Fig. 7, X40000).


sections cut parallel or obliquely to the crystal surfaces (Fig. 8A-D). When observingthe internal structure of a crystal, it usually appears either amorphous (Figs 2, 6, 7,12) or in the form of striations running parallel to the long or short axis (Figs 2, 9, 10).Less frequently, crystals appear in sections as a square lattice of electron-dense dots(Figs 4, 5, 11).

Pig. 8. A diagram to illustrate the possible three-dimensional form of the crystal (rectan-gular prism). When crystals are sectioned parallel to the AA'-BB' orBB'-CC planes, theircontours appear to be rectangular (A, B). Obliquely sectioned crystals give trapezoidal (c)and triangular (D) profiles.

Figs 9-12. A crystal tilted at various angles to the electron beam. Its internal structureappears to consist of striations running parallel to the long axis (Fig. 9, X70 000) or shortaxis (Fig. 10, X70000). It also appears as a square lattice (Fig. 11, X70 000) or amorphous(Fig. 12, X70000). The diffraction patterns are shown in the corresponding insets.

Fig. 13. A crystal in which the boxed portion is highly enlarged to demonstrate thesubstructure of the striations (cf. Figs 14, 15). X56000.

Figs 14, 15. The same highly magnified image in which striations appear as doublehelices. These helices are locally cross-bridged via short fibrils (arrowheads in Fig. 14).X1800 000.

Fig. 16. Accumulation of small blocks of aligned dots (striations), variably oriented.X 77 000.

Fig. 17. A group of well-developed crystals demonstrating a radial arrangement. Note thepresence and orientation of the striations. X46 000.

Fig. 18. Enlarged view of the edge of a striated crystal. The boundary striations appearstair-like (arrows). X74000.

Fig. 19. A cytoplasmic crystalline inclusion undergoing erosion. This can be identified inboth the outer and inner parts of the crystal (single and double arrowheads). X 108000.

Fig. 20. An ultrathin section treated with pepsin. Electron-transparent regions (asterisk)can be seen in areas of the cytoplasm where crystals have been digested. X43 000.


AFigs 9—12. For legend see p. 235.


Figs 13-15. For legend see p. 235.


Figs 16—20. For legend see p. 235.


To interpret the structure and then determine the crystal interspacing, thespecimens were rotated/tilted with respect to the electron beam and diffraction pat-terns were recorded at various angles. Thus, when a crystal is oriented in the specimenso that the electron beam becomes parallel to the crystallographic plane along its longaxis, the image shows a sequence of parallel striations that correspond to the parallelplanes (Fig. 9). The spacings between the striations are estimated to be about 100 A.Tilting and rotating the specimen until it is observed parallel to the crystallographicplanes along the short axis of the crystal, causes the orientation of the striations tochange, but the spacings remain the same (Fig. 10). When the electron beam becomessimultaneously parallel to both of these planes, the internal structure of the crystalappears as a square lattice of electron-dense dots 70-80 A in diameter, spaced at about100 A centre-to-centre (Fig. 11). Crystals oriented randomly with respect to theelectron beam appear amorphous (Fig. 12). This is due to the disappearance of thecrystallographic planes that are not in a Bragg reflection position. The diffractionpatterns of the above images are shown in the corresponding insets to the figures.

Under high magnification the striations seem to consist of double helices bridgedtogether via short fibrils (Figs 13, 14, 15). Our selection of right and left-handedhelices, illustrated in Fig. 15, was arbitrary.

The formation of the crystalline inclusions seems to start in cytoplasmic regionswhere aligned dots accumulate (Fig. 16). The latter increase further in length andbecome organized into typical crystals, which are often grouped (Fig. 17). Whenobserving the edges of striated crystal profiles they either appear sharp or demonstratea stair-like construction (Fig. 18).

In the next stage of leaf differentiation, crystalline bodies in mesophyll chloren-chyma cells appear to be undergoing erosion (Fig. 19). This process begins from theouter part of the crystal and extends progressively into the inner lumen until finallythe crystal completely disappears. This has been verified in leaves larger than 3 mm.

Identification of the proteinaceous nature of the crystals was done by treating thespecimens with pepsin. As a result electron-transparent regions were formed wherecrystals initially existed (Fig. 20).

DISCUSSION

Profiles of cytoplasmic crystalline inclusions in leaf chlorenchyma cells ofOriganum usually appear rectangular, trapezoidal and triangular. Combining theseshapes leads us to conclude that, most probably, the three-dimensional form of theseinclusions corresponds to a rectangular prism or a derivative (Fig. 8). Thus, when acrystal is sectioned parallel to the AA'-BB' or BB'-CC planes, rectangles similar tothose illustrated in Figs 4 and 5 are seen. Trapezoidal or triangular contours areobtained when crystals are obliquely sectioned (cf. Figs 6, 7).

Large or small aggregations of variously oriented aligned dots are thought torepresent initial stages of crystal formation (cf. Wrischer, 1973, for plastid in-clusions). These aligned dots later increase in length and become organized intoparallel striations. Crystals seem to grow by deposition of new striations at their edges.


This process probably does not take place simultaneously throughout the crystal, asshown in Fig. 18, where boundary striations appear unequal (one striation is overlaidby another shorter one).

High magnification of the striations showed that they consist of double helices. Thisfact is further supported by the form of the diffraction patterns recorded when thecrystal was tilted at various angles (Figs 9—11). Thus, the existence of three spots, i.e.the central one and the ± 1 order, indicates that the striations comprise a sinusoidalgrating. This implies that the density of the two groups of striations follows thesinusoidal distribution, which occurs if each striation can be projected as an ellipsoidof revolution (Moore, 1972). Since the crystals have been proved by pepsin digestionto be proteinaceous, the above-mentioned helices most probably represent proteinmacromolecules. Lawton (1978), studying the P-protein crystals in sieve elements ofpea hypocotyl, proposed a model according to which striations consist of helicallywound (perhaps double) filaments. A similar twisted form of striation was alsodescribed by Reanudin & Cheguillaume (1977), in the cytoplasmic crystalline in-clusions of Thesium humifusum haustoria. Observations made by Arsanto (1982) onthe P-protein crystals of some dicotyledons, revealed that striations are composed ofdouble helices 10—15 nm in diameter termed double helices 1 (DH 1). In cytoplasmiccrystals of O. dictamnus the double chains constituting the striations wind aroundeach other rather loosely, i.e. they appear in some sections as aligned loops (cf. Fig.15). Hence, it is suggested that the dots identified in the striations or the squarelattices correspond to the described loops, which become obvious when images aregreatly enlarged.

Striations are 70-80 A wide and are spaced about 100 A apart in both dimensions.This indicates that the arrangement of the crystallographic planes possesses tetragonalsymmetry. Several authors have measured the width and periodicity of the striationsof cytoplasmic crystalline inclusions present in both plant and animal cells. As shownin Table 1, these parameters vary over a wide range among different species. Sincesuch inclusions are generally believed to be proteinaceous (Thaler & Amelunxen,1975; Perry & Snow, 1975), the disparity in their spacing could be attributed tointernal factors determining their growth and organization (Cronshaw, Gilder &Stone, 1973).

With respect to the factors affecting crystal formation, May & Kiinkel (1975) havereported that under certain growth conditions the protein concentration of yeast cellsincreases, so that after osmotic shock crystalline bodies are formed. The latter disap-pear within 4h, provided that osmotic shock is followed by growth of the cells.Physiological stress (removal of the root system) was also considered by De Greef &Verbelen (1973) to be a cause of crystal formation. Cran & Possingham (1972) foundthat in spinach leaves when photosynthetic activity ceased crystalline inclusions wereformed in the chloroplast matrix, and disappeared after the leaves were illuminated.

Crystals may be formed in the cytoplasm as a result of a surplus protein (Yoo, 1970;Baker, Hall & Thorpe, 1978) or due to the presence of numerous ribosomes (Krishan,1970). Bennet & Smith (1979) consider that cytoplasmic crystalline bodies are com-posed of tubulin derived from disintegrated microtubules (see also De Brabander,


Table 1. Width and periodicity of the striations of cytoplasmic crystalline inclusionsin plant and animal cells

Species

Cicer arietinum(sieve elements of hypocotyl)

Dryopteris filix-mas(root apical meristem)

Gonatobotryum apiculatum(conidiogenous cell apices)

Armadillo(principal and holocrinal cells ofepididymis middle segment)

Mouse(follicular cells of thyroid)

Human(leukemic lymphoblasts)

Phaseolus multiflorusandPisum sativum(sieve elements of hypocotyl)

Human(adrenal cortex)

Pemcillium(all cells in penicillus* exceptphialides and conidia)

Chick(posterior necrotic zone ofdeveloping limb)

Human(testicular interstitial cells)

Thesium humifusum(haustoria)

Salvia glutinosa(glandular hairs of leaf and flowerbuds)

Lilium tigrinum(epidermal cells of pedicle, ovaryand leaf)

Glycine max(sieve elements of hypocotyl)

Width ofstriation

(A)100-150

35

66

50

100

105

70

Spacingsbetweenstriations

(A)100-150

100

80-100

70-90

70

250-280

120 (centralbody)

450 (tail)

76

140

530-550

150

80

105

250

115

Authors

Arsanto (1982)

Burgess (1971)

Cole (1973)

Edmonds & Nagy(1973)

Elfvin (1971)

Krishan (1970)

Lawton (1978)

Magalhaes (1972)

Metitiri & Zachariah(1972)

Mottet & Hammar(1972)

Nagano & Ohtsuki(1971)

Renaudin &Cheguillaume (1977)

Schnepf(1971)

Thaler & Amelunxen(1975)

Wergin & Newcomb(1970)


Aerts, Van de Veire & Borgers, 1975). According to Newcomb (1967) andDeshpande(1974), coated vesicles, presumably originating from the Golgi apparatus or theendoplasmic reticulum, may also contribute to the formation of P-protein crystals insieve elements. Crystalline inclusions have also been identified in tissues infected byviruses (Hampton, Phillips, Knesek & Mink, 1973; Brunt & Stace-Smith, 1978).

The erosion often observed in the cytoplasmic crystals of young Origanummesophyll cells indicates that protein exists in a storage form and is used later by thedeveloping cells (see also Lyshede, 1980). Ross, Thorpe &Costerton (1973), studyingtobacco callus cultures, speculated that disintegration of the cytoplasmic crystalsresults in the release of amino acids that were used for further synthetic processes inthe meristem. It is possible that kinetin plays an important role during this process(Jakubek & Mlodzianowski, 1978).

The authors thank Professor I. Tsekos for critically reading the manuscript and the 'StiftungVolkswagenwerk' and the National Research Foundation for financial support.

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(Received 18 April 1983 -Accepted 25 May 1983)

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ULTRASTRUCTURE, ORGANIZATION AND CYTOCHEMISTRY …For light microscopy, semithin sections of plastic-embedded tissue were staine 1d wit % Toluidinh e Blue 0 in 1 % borax solution