A CRYSTALLINE INCLUSION IN SIEVE ELEMENT NUCLEI OF AMSINCKIA

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1 J. Cell Sci. 38, 1-10 (1979) Printed in Great Britain Company of Biologists Limited A CRYSTALLINE INCLUSION IN SIEVE ELEMENT NUCLEI OF AMSINCKIA I. THE INCLUSION IN DIFFERENTIATING CELLS KATHERINE ESAU AND ANDREW C. MAGYAROSY Department of Biological Sciences, University of California, Santa Barbara, California 93106, U.S.A., and Department of Cell Physiology, University of California, Berkeley, California 94720, U.S.A. SUMMARY The presence of usually single, elongated, compound crystalloids in nuclei of sieve elements is an outstanding characteristic of the phloem of Amsinckia douglasiana A. DC. (Boraginaceae). The crystalloid consists of two components forming alternating panels, or blocks, that extend through the entire length of the crystalloid and radiate from the centre where one of the components predominates. Three to seven panels for each component were recorded. One component consists of 4-sided tubules closely packed in highly ordered aggregates, the other of wider 6-sided tubules rather loosely arranged in paracrystalline aggregates. The crystalloid arises at the beginning of sieve element differentiation. Aggregates of 4-sided tubules appear first. In plants infected with the curly top virus, the crystalloids do not differ from those in noninfected controls in structure and conformation. But because the phloem in infected plants is hyperplastic, with most of the cells differentiating as sieve elements, the crystalloids are far more abundant in diseased than in healthy plants. INTRODUCTION Ultrastructural research has considerably elucidated the structure of the foodconducting cell in the phloem, the sieve element (Evert, 1977). Electron microscopy has served to confirm, extend, and modify concepts based on light microscopy and has disclosed details that bear on the functional specialization of the cell. Many ultrastructural features of the sieve element are common to taxa of diverse evolutionary levels. Only a few appear to be restricted in distribution and may be of systematic value. Behnke (1977) has successfully utilized the presence or absence of protein inclusions in the sieve element plastid for delimitation of higher taxa of flowering plants. Another differentiating feature, still hardly examined for its taxonomic value, is the paracrystalline protein that is sporadically encountered in sieve element cytoplasm in dicotyledons (Esau, 1978). Differences in occurrence and conformations of P-protein (Cronshaw, 1974; Evert, 1977) may be significant with regard to the evolutionary specialization of sieve elements as conducting cells. In the present paper we report the occurrence of a characteristic paracrystalline nuclear inclusion in sieve elements of Amsinckia douglasiana A. DC. (Boraginaceae). Nuclear inclusions are common in vascular plants at all levels of specialization, but rarely have they been reported in sieve elements (see Discussion). The nuclear

2 2 K. Esau and A. C. Magyarosy inclusion in Amsinckia is a large compound crystalloid composed of 2 contrasting aggregations of subunits. If found in other Boraginaceae, and perhaps in some related families as well, this striking type of crystalloid could serve as a valuable systematic character. Beside the possible comparative value of the Amsinckia type of nuclear crystalloid, the destiny of the inclusion during cell maturation is of considerable interest in view of the removal of the nucleus and other changes in the protoplast when the cell differentiates into a conduit of photosynthates. The effect of nuclear breakdown on the crystalloid is considered in the second of 2 papers dealing with the nuclear inclusion in the sieve element of Amsinckia (Esau & Magyarosy, 1979&). The crystalloids of Amsinckia douglasiana were observed in connexion with studies of the effect of infection with the curly top virus on the phloem of this plant (Esau & Magyarosy, 1979 a). The occurrence of nuclear inclusions in sieve elements facilitated the delimitation of hyperplasia in the phloem, a symptom characteristic of curly top diseased plants (Esau, 1976; Esau & Hoefert, 1978). It also served to document nuclear disintegration in a maturing sieve element, a phenomenon the validity of which as a characteristic step in the ontogeny of the cell is sometimes questioned. MATERIALS AND METHODS Seedlings of Amsinckia douglasiana A. DC. were raised in soil in a greenhouse and were infected with the curly top virus by allowing viruliferous leaf hoppers, Circulifer tenellus (Baker), to feed on one of the primary leaves for 5 days. Leaves with pronounced symptoms of the disease were collected for electron-microscopic studies. Leaves from non-inoculated control plants grown in the same greenhouse were also collected. (Cells and tissues of non-infected, symptom-free plants are called normal for convenience.) The study was carried out on the phloem of minor veins. Previous experience with infections with curly top and other phloemlimited viruses has shown that minor veins are a particularly convenient tissue region for finding the virus and observing the effects of the infection on the phloem tissue. Leaf blade pieces containing minor veins were fixed for 2 h in 4% glutaraldehyde in o-i M sodium cacodylate buffer (ph 6'8). After a brief rinse in the buffer the material was post-fixed for 1 h in cold, buffered 2 % osmium tetroxide. A rapid evacuation in 2,2-dimethoxy propane was followed by embedment in Spurr's (1969) epoxy resin. Sections were stained on the grids with uranyl acetate and lead citrate. Fig. 1. Transection of part of vascular bundle of a minor vein from a non-infected leaf. Large cells above, part of bundle sheath of phloic origin. Obliterated early sieve elements and companion cells at ob. Central region, phloem with sieve elements (circles) and companion cells (triangles). One differentiating sieve element shows a crystalloid (cr). Lateral sieve plate at sp. Xylem elements at*. These are separated from the phloem by vacuolated parenchyma cells with chloroplasts. x Fig. 2. Transection of part of vascular bundle of a minor vein from a leaf infected with curly top virus. Underneath the bundle sheath cells (above) is hyperplastic phloem, which extends to the xylem (x). It contains mature and differentiating sieve elements (circles) and a few companion cells (triangles). Mature sieve elements are clear, the differentiating ones have dense cytoplasm and many show nuclear crystalloids. Partly obliterated sieve element at ob. x 2100.

3 Crystalloids in nuclei of Amsinckia, I

4 K. Esau and A. C. Magyarosy OBSERVATIONS Normal and hyperplastic phloem Cell division is of limited duration in minor veins of healthy leaves. In a nearly mature minor vein of medium size (Fig.

4 4 K. Esau and A. C. Magyarosy OBSERVATIONS Normal and hyperplastic phloem Cell division is of limited duration in minor veins of healthy leaves. In a nearly mature minor vein of medium size (Fig. i), a layer of thin-walled, highly vacuolated parenchyma with chloroplasts, one to two cells deep, separates the phloem from the xylem (x). The phloem forms a layer two to three cells deep and contains sieve elements (circles) with companion cells (triangles) and a few parenchyma cells that are wider than either of the 2 other kinds of cells. On the outer periphery of the phloem are rather large parenchyma cells with thickened walls. These cells are part of the oldest phloem in which the earliest sieve elements and companion cells have become obliterated (Fig. 1, ob). In Fig. 1, all sieve elements shown are mature except one, in which a nuclear crystalloid (cr) is seen in transection. A nuclear envelope delimits the nucleus with the crystalloid but is too faint to be discerned at the low magnification in that figure. In minor veins of curly top diseased leaves, cell division continues longer than in non-infected leaves and results in the production of a hyperplastic phloem that is more massive than the normal. In the hyperplastic phloem, as shown in Fig. 2, the majority of cells are sieve elements (circles), mature or immature. Despite the large volume of sieve elements, companion cells (triangles) are scarce, for many sieve elements have no companion cells. The abnormal phloem is four to five cells deep and is not separated from the xylem (x) by thin-walled vacuolated parenchyma cells. Differentiating phloem cells occur next to the xylem. As in normal phloem, the mature sieve elements in the hyperplastic phloem contain little stainable material, whereas the immature cells have relatively dense cytoplasm. Several mature sieve elements appear in a group to the left in Fig. 2. Many of the immature sieve elements in Fig. 2 show nuclei with crystalloids. The sieve elements of the hyperplastic phloem vary more in width, and form a less-regular tissue, than do sieve elements of normal phloem. The hyperplastic phloem cells also have thicker than normal cell walls. Parenchyma cells in the affected phloem have denser cytoplasm and those located on the outer periphery of the vein have conspicuously thicker cell walls than the corresponding cells in the normal phloem. Both kinds of parenchyma cells, those among the sieve elements and those on the periphery of the phloem, may contain virus particles (Esau & Magyarosy, 1979 a). Figs Cells and parts of cells from differentiating hyperplastic phloem of an infected leaf. The components of crystalloids are marked ex and cy. Fig. 3. Nucleus with 2 crystalloids in transection. x Fig. 4. Nuclear crystalloid in transection. x Fig. 5. Nuclear crystalloid in longitudinal section, x Inset: transection of part of crystalloid showing compactly arranged ex tubules with square transections (right and left) and more loosely arranged cy tubules with hexagonal transections (middle), x Fig. 6. A group of 3 differentiating sieve elements in transection. Mature crystalloid in the nucleus of the cell to the right. Beginning of crystalloid formation in the cell to the left: a small aggregate of ex is next to the nucleolus (see Fig. 7). x

5 Crystalloids in nuclei of Amsinckia, I

6 6 K. Esau and A. C. Magyarosy Despite the differences between normal and hyperplastic phloem in tissue development, cellular composition, and form and arrangement of cells, the differentiation of sieve elements in the infected phloem follows the same sequence as it does in normal tissue. An immature sieve element has a protoplast with components that are common in young plant cells and, during maturation, the cell becomes devoid of nucleus and ribosomes, and its tonoplast breaks down. The endoplasmic reticulum becomes agranular and passes through the characteristic transitory stage during which it is stacked. Starch-depositing plastids and mitochondria are retained. Sieve plates and lateral sieve areas have the structure common in dicotyledons, and branched plasmodesmata connect sieve elements and companion cells. The developmental events, including the appearance and subsequent changes of the nuclear crystalloids, are more readily followed in the hyperplastic phloem than in the normal because of the large volume of simultaneously differentiating sieve elements in the former (Fig. 2). In this and in the companion paper on crystalloids in Amsinckia (Esau & Magyarosy, 19796), both normal and hyperplastic sieve elements are used to describe the nuclear crystalloids and their changes during cell development and maturation. Form and development of nuclear crystalloids Most sieve element nuclei contain one crystalloid (Figs. 2, 6); occasional ones have two (Fig. 3) or a double crystalloid (Fig. 8). The nucleus and its crystalloid are elongated parallel to the long axis of the cell. Hence, a longitudinally sectioned cell exposes the crystalloid in longitudinal view (Fig. 5). The crystalloid is compound, for it consists of 2 contrasting components. Panels, or blocks, in which the subunits form dense, highly ordered aggregates, alternate with usually narrower panels in which the subunits are less orderly and more loosely aggregated (Fig. 4). The panels radiate from a centre in which the dense component predominates but is intermingled with some elements of the less-dense component. To simplify further description of the crystalloid, the dense component is designated ex (crystalloid component x), the loose component cy (crystalloid component y). Figs Transactions of cells from infected (Figs. 7-9, 11) and non-infected (Figs. 10, 12) leaves. The components of the crystalloids are marked ex and cy. Fig. 7. Nucleolus and ex aggregate from the nucleus shown in Fig. 6. x Fig. 8. Nucleus with a nucleolus and double crystalloid in transection. Crystalloid consists of 2 components but 2 small aggregates of cy are free in the nucleoplasm. x Fig. 9. Differentiating sieve element with nucleus including a nucleolus and 2 ex aggregates (see Fig. 11). x Fig. 10. A group of 3 cells: below, parenchyma cell; above, sieve element with nucleus (right) and companion cell (left). The nucleus contains 2 aggregates, x Fig. 11. Two ex aggregates from the nucleus shown in Fig. 9. The section passed parallel to the tubules in the aggregate to the left and at various angles to the tubules in the aggregate to the right, x Fig. 12. Two ex aggregates from another section of the sieve element nucleus shown in Fig. 10. x

7 Crystalloids in nuclei of Amsinckia, I

8 8 K. Esau and A. C. Magyarosy Both components consist of tubules longitudinally oriented in the crystalloid. In the wider cy tubules, the electron-lucent lumina are more clearly discernible than in the narrower ex tubules. The transections of the individual ex tubules are square, those of the cy tubules are hexagonal (Fig. 5, inset). In other words, the ex tubules are 4-sided and the cy tubules are 6-sided. The 4-sided tubules are much more closely packed than are the 6-sided tubules. In biased sections, the ex and cy tubules are seen to be continuous with one another at the interfaces between the ex and cy panels, with a transition in form between the 2 kinds of tubules (Fig. 4; also fig. 8 in Esau & Magyarosy, 19796). The 6-sided tubules appear to be interconnected with each other by extensions from the angles (Fig. 5, inset). Nuclei, or parts of nuclei, with paracrystalline aggregates in early stages of development are depicted in Figs. 6 (cell to the left), 7, and Parts of nuclei shown in Figs. 6 and 9 appear enlarged in Figs. 7 and 11, respectively. Fig. 12 illustrates the same paracrystalline aggregates as Fig. 10 but taken at higher magnification and from another section of the same cell group. In Figs. 7 and 9-12, the developing crystalloids are ex. It appears, therefore, that the ex form of crystalloid is organized before the cy form. In Fig. 8, in which ex and some cy are seen assembled into a double crystalloid, 2 cy aggregates occur separately in the nucleoplasm. In early stages of crystalloid development, ex also may be found in separate aggregates in one nucleus (Figs. 9-12). Since later the paracrystalline matter usually appears to be assembled into one or two compound crystalloids (Figs. 3, 4, 6), union of separately formed aggregates may be one of the developmental steps in the crystalloid formation. The crystalloid develops in the presence of nucleolus (Figs. 6-9). Sometimes the forming crystalloid is so close to the nucleolus that the latter has a concavity facing the crystalloid (Figs. 6, 7). The nucleolus is still present and shows no decrease in size when the crystalloid assumes mature size and form (Fig. 8). Chromatin is remarkably sparse in younger and older nuclei and is limited to small aggregates near the nuclear envelope (Figs. 2, 3, 6, 9, 10). The group of 3 cells from a non-infected leaf depicted in Fig. 10 demonstrates a significant aspect of crystalloid development. The cell group was formed by 2 divisions, the first resulting in the formation of the lower cell and the precursor of the 2 cells above, the second producing the cell wall (w) between the 2 upper cells. The thinness of the second wall indicates that the division that produced it occurred a short time before the tissue was fixed for microscopy. The nucleus of the larger of the 2 upper cells contains 2 ex aggregates (Fig. 12). Hence the cell may be interpreted as a young sieve element. The cell to its left is probably a companion cell. It thus appears that the formation of nuclear crystalloids is an early event in the differentiation of a sieve element and identifies the cell possibly directly after the latter is formed by division of a phloem mother cell. DISCUSSION Reviews by Thaler (1966) and Wergin, Gruber & Newcomb (1970) indicate that nuclear inclusions, that is, aggregates of matter apparently not nucleolar or hetero-

9 Crystalloids in nuclei of Amsinckia, I 9 chromatic in nature, are wide-spread in the plant kingdom. Most studies of such inclusions were made with light microscopes, but lately some electron-microscope investigations have been carried out (e.g. Wergin et al. 1970). Many nuclear inclusions show a smaller or greater degree of crystallinity, others are described as fibrous, granular, or amorphous, that is with poorly defined substructure. Those inclusions that were tested for their chemistry were found to be proteinaceous. Inclusions may consist of virus particles, some in paracrystalline arrays (e.g. Esau & Hoefert, 1972; Weintraub & Ragetli, 1970). Nuclear inclusions commonly occur in basically parenchymatous types of cells but located in a variety of organs and tissues. The inclusions may be limited to a certain type of tissue or even to a certain type of parenchymatous cell (Wergin et al. 1970). References to the presence of nuclear inclusions in sieve elements are relatively few. In sieve elements of Tilia americana, Evert & Deshpande (1970) found aggregates of P-protein with tubular components of the same kind as were present in the cytoplasm. Esau (1978) recorded fibrous aggregates, with components resembling extended P- protein tubules, in sieve element nuclei of Gossypium hirsutnm. Hebant (1969), using a light microscope, observed nuclear crystalloids in sieve elements, as well as other cell types, in several ferns. Evert & Eichhorn (1974) studied nuclear crystalloids of a fern {Platycerium bifurcation) sieve elements with an electron microscope and found them to be elongated bodies oriented parallel or at an angle to the long axis of the nucleus. This crystalloid gave a positive reaction for protein with mercuric bromphenol blue stain and was composed of tubular elements of considerable length. Similar crystalloids were found in pericyclic (parenchymatous) cells. The nuclear crystalloids of Amsinckia, which were seen only in sieve elements, differ from those in Platycerium in being compound but resemble them in having tubular subunits. The close packing of tubules in Platycerium crystalloids is comparable to that in the ex aggregate of Amsinckia crystalloid, but the ex tubules are 4-sided whereas those in Platycerium are probably 6-sided as suggested by a highmagnification view of the fern crystalloid in transection. Some authors mention close spatial relation between nucleoli and nuclear inclusions and suggest a developmental relation between the 2 structures, which may involve a diminution of nucleolar size (cf. Thaler, 1966). Wergin et al. (1970) observed extensive contact between nucleoli and nuclear inclusions in some species. In ferns, these authors also found that nuclei of young parenchyma cells had nucleoli but no inclusions, those of older cells had inclusions but rarely showed nucleoli. In Amsinckia, crystalloids in early stage of formation were seen close to the nucleoli but the latter did not appear to change when the crystalloids were no longer growing. In curly top diseased plants, nuclear crystalloids are more abundant than in the healthy plants simply because the hyperplastic phloem of infected plants contains many more sieve elements than the normal. There is no direct relation between infection and crystalloid formation. Presence of crystalloids in non-infected control plants of Amsinckia and their absence in curly top infected Beta and Spinacia (Esau, 1976; Esau & Hoefert, 1978), which normally have no nuclear crystalloids in sieve elements, support this conclusion.

10 io K. Esau and A. C. Magyarosy As was suggested in the Introduction, it would be worthwhile to determine whether nuclear crystalloids of the type found in Amsinckia occur in sieve elements of other Boraginaceae and related families. In view of their large size and characteristic form these crystalloids may prove to be a useful systematic character. The research for this paper was supported in part by U.S.A. National Science Foundation Grant PMC REFERENCES BEHNKE, H.-D. (1977). Transmission electron microscopy and systematics of flowering plants. Plant Syst. Evol., Suppl. 1, CRONSHAW, J. (1974). P-proteins. In Phloem Transport (ed. S. Aronoff, J. Dainty, P. R. Gorham, L. M. Srivastava & C. A. Swanson), pp New York and London: Plenum Press. ESAU, K. (1976). Hyperplastic phloem and its plastids in spinach infected with the curly top virus. Ann. Bot. 40, ESAU, K. (1978). The protein inclusions in sieve elements of cotton (Gossypium hirsutum L). J. Ultrastruct. Res. 63, ESAU, K. & HOEFERT, L. L. (1972). Infrastructure of sugarbeet leaves infected with beet western yellows virus. J. Ultrastruct. Res. 40, ESAU, K. & HOEFERT, L. L. (1978). Hyperplastic phloem in sugarbeet leaves infected with the beet curly top virus. Am. J. Bot. 65, ESAU, K. & MAGYAROSY, A. C. (1979a). Nuclear abnormalities and cytoplasmic inclusion in Amsinckia infected with curly top virus. J. Ultrastruct. Res. 66, ESAU, K. & MAGYAROSY, A. C. (19796). Crystalline inclusion in sieve element nuclei of Amsinckia II. The inclusion in maturing cells. J. Cell Set. 38, EVERT, R. F. (1977). Phloem structure and histochemistry A. Rev. PI. Physiol. 28, EVERT, R. F. & DESHPANDE, B. P. (1970). Nuclear protein in sieve elements of Tilia americana. J. Cell Biol. 44, EVERT, R. F. & EICHHORN, S. E. (1974). Sieve-element ultrastructure in Platycerium bifurcation and some other polypodiaceous ferns: the nucleus. Planta 119, HEBANT, C. (1969). Observations sur le phloeme de quelques Filicinees tropicales. Natttralia monospeliensia. Se'r. Bot. 20, SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, THALER, I. (1966). Eiweisskristalle inpflanzenzellen. Protopiasmatologia II Iteby, 87 pp. WEINTRAUB, M. & RAGETLI, H. W. J. (1970). Electron microscopy of the bean and cowpea strains of Southern bean mosaic virus within leaf cells. J. Ultrastruct. Res. 32, WERGIN, W. P., GRUBER, P. J. & NEWCOMB, E. H. (1970). Fine structural investigations of nuclear inclusions in plants. J. Ultrastruct. Res. 30, {Received 10 October 1978)

Parenchyma Cell. Magnification 2375X

Parenchyma Cell. Magnification 2375X Parenchyma Cell The large size of parenchyma cells is due in part to their relatively large vacuole (V) and in part also to the large number of chloroplasts (Cp) they contain. From a crimson clover, Trifolium