Planta (Berl.) 95, 281~296 (1970) 9 by Springer-Verlag 1970
On the Occurrence of Nuclei in Mature Sieve Elements* RAY F. EV~RT, JEI~RY D. DAvls, C. MILTON Tucl;~a and FRANK J. ALFIERI Department of Botany, University of Wisconsin, Madison l~eceived July 23 / September 18, 1970 Summary. The secondary phloem of 3 species of the Taxodiaccae and 13 species of woody dicotyledons was examined for the occurrence of nuclei in mature sieve elements. Nuclei were found in all mature sieve cells of Metasequoia qlyptostroboides, Sequoia sempervirens and Taxodium distiehum, and in some mature sieve-tube members in 12 of the 13 species of woody dicotyledons. Except for nuclei of sieve cells undergoing cessation of function, the nuclei in mature sieve cells of M. glyptostroboides, S. sempervirens and T. distiehum were normal in appearance. The occurrence and morphology of nuclei in mature sieve-tube members of the woody dicotyledons were quite variable. Only 3 species, Robinia pseudoaeacia, Ulmus americana and Vitis riparia, contained some mature sieve elements with apparently normal nuclei. Introduction P h l o e m i n v e s t i g a t o r s g e n e r a l l y agree t h a t t h e sieve e l e m e n t lacks a nucleus a t m a t u r i t y . E a r l y accounts of t h e presence of nuclei in m a t u r e sieve elements (Fischer, 1886 ; Lecomte, 1889 ; S c o t t a n d Brebner, 1889 ; S c h m i d t , 1917) were g e n e r a l l y dismissed as m i s i n t e r p r e t a t i o n s . H o w e v e r , during t h e p a s t few y e a r s new r e p o r t s h a v e been m a d e of t h e presence of nuclei in some m a t u r e sieve elements in a wide v a r i e t y of v a s c u l a r p l a n t s , including Isoetes (Paolillo, 1963), several species of conifers ( E v e r t a n d Alfieri, 1965; M u r m a n i s a n d E v e r t , 1966; S r i v a s t a v a a n d O'Brien, 1966; W o o d i n g , 1966), Austrobaileya ( S r i v a s t a v a , 1970), Neptunia (Shah a n d J a m e s , 1968), Ulmus ( E v e r t et al., 1969), Secale ( 0 ' B r i e n a n d Thim a n n , 1967), Smilax (Ervin a n d E v e r t , 1970) a n d some of t h e p a l m s ( P a r t h a s a r t h y , 1966). I n m o s t cases these nuclei were described as being d e g e n e r a t e d or necrotic in a p p e a r a n c e . Nuclei of n o r m a l a p p e a r a n c e or m o r p h o l o g y were e n c o u n t e r e d in m a t u r e sieve elements in o n l y two of t h e species i n v e s t i g a t e d , Smilax hispida (Ervin a n d E v e r t , 1970) a n d Ulmus americana ( E v e r t et al., 1969). N o n e of the r e c e n t i n v e s t i g a t i o n s cited a b o v e was u n d e r t a k e n p r i m a r i l y to d e t e r m i n e t h e occurrence of nuclei in m a t u r e sieve elements. Because * This research has been supported by National Science Foundation grants GB-5950 and GB-8330. 19 Planta (Berl.), Bd. 95
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Table. Occurrence and morphology o] nuclei in mature sieve elements Species
Occurrence
Morphology
Metasequoia glyptostroboides Hu and Cheng Sequoia sempervirens (D. Don) Endl.
In all elements In all elements In all elements Occasional
Normal
Taxodium distichum (L.) Rich. Acer negundo L. Acer saccharinum L. Comus racemosa Lam. Comus stoloui/era Miehx. Juglans nigra L.
Occasional Frequent Occasional to frequent Occasional
Populus tremuloides Michx.
Occasional
Quercus alba L. Rhus glabra L.
Occasional Occasional
Robinia pseudoacacia L.
Frequent
Salix nigra Marsh. Tilia americana L. Ulmus americana L.
None Occasional Occasional to frequent
Vitis riparia Michx.
Occasional
Normal Normal Clear to dense spherical body Clear spherical body Clear spherical body Dense spherical to elongate body Relatively dense spherical body Relatively clear spherical body, sometimes swollen Clear spherical body Clear to dense spherical body Normal to dense spherical body Clear crumpled body Normal to clear spherical body, sometimes swollen Normal to clear spherical body
of the recent reports of nuclei in mature sieve elements and of the importance of this feature in the overall understanding of sieve-element ontogeny, structure and function, the present investigation was undertaken. The species of woody dicotyledons selected for investigation (Table) were largely those already chosen for studies of seasonal phloem development and for which extensive, year-round collections of tissues were available. Three conifers, Metasequoia gIyptostroboides, Sequoia sempervirens and Taxodium distichum, all members of the Taxodiaceae, were also examined.
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Metasequoia was of special interest, for its sieve cells have been studied intensively a t the ultrastructural level b y Kollmann a n d Schumacher (1964, and literature cited therein). I n a review article, Kollmann (1964) reported t h a t a n u c l e u s - - " t h o u g h of changed shape " - - c o u l d still be found in sieve cells with well developed connections. Although he reported briefly on the process of nuclear disintegration in Metasequoia sieve cells, he did not state at what age or stage of sieve cell developm e n t t h a t process took place. One of the goals of the present investigation was to answer t h a t question.
Materials and Methods The Metasequoia glypto~troboides material was obtained from 2-to-5-year-old shoots of trees grown in the greenhouse and out-of-doors in Madison, Wisconsin. Two to four trees were sampled on each of 16 collection dates over a 3-yearperiod. Tissues were collected on the following dates: 29 J a n u a r y 1966, 2 F e b r u a r y 1967, 14 and 20 March 1966, 9 a n d 20 April 1965, 8 and 20 April 1966, 5 April 1967, 29 May 1966, 18 a n d 24 J u n e 1965, 17 J u n e 1966, 1 J u l y 1966, and 5 and 29 November 1966. The Sequoia sempervirens material was collected on 15 November 1967, through the courtesy of the Crown Simpson Corporation, San Francisco, California. The Taxodium distichum material was collected on 28 August 1967, at Henderson, Tennessee. Tissues of b o t h were obtained from the trunks of apparently healthy trees. Most tissues were immersed immediately in formaldehyde-acetic acid-alcohol (FAA; Sass, 1958, p. 15) and shortly afterward aspirated in the laboratory. A]] tissues were embedded in celloidin and sectioned with a sliding microtome a t 15 ~z. Serial transverse, radial and tangential sections were obtained of all collections. The sections were tied onto slides with thread, stained with tannic acid-ferric chloride-resorcin blue (Cheadle et al., 1953), and then mounted in Harleco synthetic resin. Seasonal collections were available of all woody dicotyledons (Table) except Robinia. Tissues were obtained from the trunks of apparently healthy trees 7 or more years old growing in the Eagle Heights area of the University of Wisconsin campus and in the University of Wisconsin Arboretum at Madison. Two to four trees were sampled a t each collection date. The n u m b e r of collection dates ranged from 14 over a I-year-period for Cornus racemosa, Juglans nigra, Salix nigra and Ulmus americana to 64 over a 2-year-period for Acer negundo, Rhus glabra and Tilia americana. All months of the year were represented. Collection procedures were the same as those outlined by Tucker and Everg (1969) for a study of seasonal phloem development in Acer negundo. The materials were placed in either chromic acid-acetic aeid-formMdehyde (Craf I I I ; Sass, 1958, p. 18) or F A A in the field a n d aspirated shortly afterward in the laboratory. Serial transverse and radial sections were obtained of all collections, and serial tangential sections of selected ones. As with the conifer sections, these were stained by the method of Cheadle et al. (1953). The Vitis tissues were the same as those used in a recent study of seasonal phloem development in vines (Davis and Evert, 1979). I n addition to the seasonal collections, other tissues of the dicotyledonous species (with the exception of Juglans nigra, Rhu8 glabra and Viti8 r@aria) were collected during the summer of 1967, on the following dates: 8 and 19 June, 10 July, 9 August, a n d 18 September. All these tissues, including those of Robinia, were treated in the variety of ways outlined for the tissues of Ulmus collected on the same dates in 1967, as reported in a recent article (Evert et al., 1969). I n addition, as with the Ulmus study, examination of fixed materials was supplemented with t h a t of fresh material. 19"
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t~. F. Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 1--4
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Results Taxodiaceae. The s e c o n d a r y p h l o e m of Metasequoia (Figs. 1-6), Sequoia (Figs. 7-9) a n d Taxodium (Figs. 10-12) is v e r y similar in composition a n d a r r a n g e m e n t . The axial s y s t e m consists of fibers, sieve cells, s t r a n d s of p a r e n c h y m a cells, a n d a l b u m i n o u s cells. The m o s t l y uniseriate r a y s consist o5 o r d i n a r y r a y p a r e n c h y m a cells a n d a l b u m i n o u s cells, t h e l a t t e r g e n e r a l l y occurring on t h e m a r g i n s of t h e rays. Cells of t h e axial s y s t e m a l t e r n a t e r a d i a l l y in a sequence of sieve cells, p a r e n c h y m a cells, sieve cells, fibers, sieve cells, p a r e n c h y m a cells, a n d so on (Figs. 1, 2, 5, 6, 10, 11). Local irregularities do occur, however, a n d are m o s t c o m m o n n e a r t h e ends of t h e g r o w t h increments. The fibers of the e a r l y p h l o e m are c o m m o n l y wider a n d h a v e t h i c k e r walls t h a n those of the ]ate p h l o e m ; therefore, g r o w t h i n c r e m e n t s generally are easily d e l i m i t e d in all t h r e e species. This f e a t u r e was of g r e a t value in t h e i n t e r p r e t a t i o n of t h e sequence of events in t h e d e v e l o p m e n t of the s e c o n d a r y p h l o e m of Metasequoia a n d in t h e d e t e r m i n a t i o n of t h e a p p r o x i m a t e ages of t h e sieve cells in all t h r e e species. Mature sieve cells were identified through the presence of fully-developed sieve areas and of crystals in the radial walls. As in Pinus (Evert and Alfieri, 1965), the sieve-area pores in Metasequoia, Sequoia and Taxodium occur in groups. AIthough individual pores are difficult to detect in living sieve cells with the light microscope, fully-developed pore groups are detectable, if only with aid of an oilimmersion objective. Unlike their counterparts in the Pinaceae, mature sieve cells in the Taxodiaceae lack secondary walls. All mature cell types in the secondary phloem of the species of Taxodiaeeae examined during the present study are characterized by the presence of numerous, small crystals in the middle lamella of the radial walls, a phenomenon reported earlier for the secondary phloem of Juniperus (Evert and Alfieri, 1965). The crystals, which are absent at the sieve areas and primary pit-fields, are lacking in cambial zone cells and in immature ceils adjacent to the cambium. Although the present article is not concerned with the seasonal cycles of phloem development of the species examined, a thorough understanding of the nuclear condition of the sieve cells in Metasequoia is virtually impossible without certain aspects of its seasonal cycle of phloem development first being understood. The seasonal cycle of phloem development of Metasequoia is, in some respects, similar to that of Pinus (Alfieri and Evert, 1968). In both, all hut the last-formed sieve
Figs. 1-4. Metasequoia glyptostroboides. Fig. 1. Transection showing some xylem, the dormant cambial zone, and mostly nonfunctional phloem. Unlabeled arrows point to nuclei in mature sieve cells. Collected 5 November 1966. • 610. Fig. 2. Radial section showing some xylem, the dormant cambial zone, some functional and nonfunctional phloem. Unlabeled arrows point to sieve areas containing variable amounts of eallose. Collected 5 November 1966. • Figs. 3, 4. Radial and tangential sections, respectively, showing nuclei (unlabeled arrows) in mature, overwintering sieve cells. Collected 5 April 1967. • CZ cambial zone; F fiber; P parenchyma cell; /~ ray; S sieve cell; X xylem
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1~. F. Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 5--9
Nuclei in Mature Sieve Elements
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cells cease functioning the same year they are derived from the cambium. The former overwinter and most remain functional until new sieve ceils differentiate in spring. The first new sieve cells of spring develop directly from cells which overwintered in an undifferentiated state on the outer margin of the cambial zone. At about the same time, or shortly afterward, eambial activity is resumed and new phloem initials are produced. In Pinus, the sieve cells that overwinter in a mature state generally do not become nonfunctional until after early phloem differentiation is completed in late May or June in the Madison, Wisconsin area. In Mstasequoia, cessation of function of the overwintering sieve cells occurs much earlier, being initiated in some cells before, and in others shortly after the initiation of new sieve cell differentiation in April. Figs. 1 a n d 2 i l l u s t r a t e t h e condition of the c a m b i a l zone a n d t h e c u r r e n t y e a r ' s p h l o e m i n c r e m e n t in a N o v e m b e r collection of Metasequoia. E x c e p t for two to t h r e e layers of sieve cells n e a r e s t t h e c a m b i u m , t h e sieve cells c o n t a i n e d conspicuous a m o u n t s of definitive eallose (Fig. 2). The sieve cells f a r t h e s t from t h e c a m b i u m - - t h o s e containing the g r e a t e s t a m o u n t s of c a l l o s e - - w e r e a l r e a d y nonfunctional. Those n e a r e s t t h e cambium, t h e last two to t h r e e layers which c o m m o n l y overwinter in a f u n c t i o n a l state, c o n t a i n e d p r o t o p l a s t s w i t h nuclei of n o r m a l a p p e a r a n c e (Fig. 1): The p r o t o p l a s t s of sieve cells between these two e x t r e m e s were in v a r y i n g stages of d e g e n e r a t i o n a n d c o n t a i n e d nuclei more or less necrotic in a p p e a r a n c e . I n all w i n t e r collections t h e two to three layers of overwintering, m a t u r e sieve cells c o n t a i n e d nuclei which were n o r m a l in a p p e a r a n c e . I n a d d i t i o n , their c o u n t e r p a r t s in t h e A p r i l collections also c o n t a i n e d such nuclei (Figs. 3, 4), e x c e p t in cells undergoing cessation of function a t t h a t time. Fig. 5 is a view of the c a m b i a l region a n d a d j a c e n t s e c o n d a r y p h l o e m of a n o t h e r A p r i l collection. H e r e t h e o u t e r m o s t l a y e r of m a t u r e sieve cells c o n t a i n e d s u b s t a n t i a l a m o u n t s of definitive callose, while t h e l a y e r n e a r e s t t h e c a m b i u m h a d v e r y little. The u n l a b e l e d arrow p o i n t s to a l a y e r of u n d i f f e r e n t i a t e d cells which, j u d g i n g from other collections, n o r m a l l y would h a v e d e v e l o p e d d i r e c t l y into t h e first, new sieve cells of spring.
Figs. 5 and 6. Metasequoia glyptostroboides. Fig. 5. Transection showing some xylem, the dormant cambial zone, two bands of mature, overwintering sieve cells, and nonfunctional phloem. Definitive callose (C) has begun to accumulate in outermost band of overwintering sieve cells. Unlabeled arrow points to layer of probable sieve-cell primordia. Collected 20 April 1966. • 400. Fig. 6. Radial section showing nuclei (unlabeled arrows) in mature sieve cells. Collected 24 June 1965. X400 Figs. 7-9. Sequoia sempervirens. Fig. 7. t~.adial section showing nuclei in mature sieve cells farthest from the cambium. Figs. 8, 9 (radial and tangential sections, respectively). Normal-appearing nuclei (unlabeled arrows) in mature sieve cells. X440. C callose; CZ cambial zone; F fiber; P parenchyma cell; S sieve cell
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Figs. 10-12. Taxodium distichum. Fig. 10. Transection showing cambial zone and mostly functional phloem. Unlabeled arrow points to nucleus in mature sieve cell. X250. Figs. 11, 12. Radial sections showing nuclei (unlabeled arrows) in mature sieve cells. The sieve cell in Fig. 12 was in band of sieve cells farthest from the cambium. Its nucleus is necrotic in appearance, x 400. CZ cambial zone; F fiber; P parenchyma cells; R ray; S sieve cell; S A sieve area
Figs. 13-19. Views of mature sieve-tube members with nuclei (unlabeled arrows) in longitudinal (Figs. 13-16, 18 and 19) and transverse (Fig. 17) sections. The nucleate sieve element in Fig. 16 is nonfunctional. Figs. 13, 14: Acer negundo. • 640. Figs. 15, 16: Acer saccharinum, x400 and • 640, respectively. Figs. 17, 18: Comus racemosa. • Fig. 19: Comus stoloni/era. • CZ cambia] zone; P parenchyma cell; S sieve-tube member; S P sieve plate; X xylem
Nuclei in M~turc Sieve Elements
Figs. 13--19
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R. F, Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 20-25. Longitudinal views of mature (Figs. 20, 22-25) and immature (Fig. 21) sieve-tube members with nuclei (unlabeled arrows) Fig. 20: Juglans nigra. • 640. Figs. 21, 22: Populus tremuloides. Arrow heads point to sphericalinclusions. • 640. Fig. 23. Quercus alba. • 640. Figs. 24, 25: Rhus glabra. • 640 and • 500, respectively. S P sieve plate
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Subsequent seasonal collections showed t h a t the retention of nuclei of normal appearance until the cell begins to cease functioning, determined by the accumulation of definitive callose at the sieve areas and by degeneration of the protoplast, is not unique to overwintering sieve cells, but is common for all sieve cells in the secondary phloem of Metasequoia. Fig. 6 is a radial view of a newly-formed, although incomplete, phloem increment of a late June collection. Nuclei (arrows) can be seen in mature cells of the fourth, fifth and sixth layers of sieve cells from the cambium. These are representative of all mature, presumably functional sieve cells in the secondary phloem of Metasequoia. The Feulgen reaction was performed on several collections of Metasequoia tissue and, in each, the nuclei of all cell t y p e s - - b o t h mature and immature sieve cells, parenchyma cells, albuminous cells, and cambial cells--gave strongly positive staining reactions. All of the mature sieve cells encountered in the secondary phloem of Sequoia and Taxodium contained nuclei. In both collections only the outermost one or two layers of sieve cells--sieve cells undergoing cessation of function--contained nuclei more or less necrotic in appearance. In Sequoia the nuclei in such cells had a dense, almost amorphous appearance (Fig. 7) while in comparable cells of Taxodium the nuclear envelope appeared to be separating from the rest of the nucleus (Fig. 12). Figs. 8 to l l illustrate nuclei representative of those found in the majority of mature sieve cells in Sequoia and Taxodium. Woody Dicotyledons. The occurrence of nuclei in mature sieve-tube members (i. e., in sieve-tube members with fully-perforated sieve plates) of the 13 species of woody dicotyledons was quite variable (Table). However, nuclei were encountered in some mature sieve elements in all but one of them, namely, Salix nigra. Nuclei occurred occasionally in mature sieve elements of eight species and frequently in two others. Within still two other species, Comus stoloni/era and Ulmus americana, the occurrence of nuclei ranged from occasional in some collections to frequent in others. If nuclei are normally absent from mature sieve elements of any of the species examined, they apparently do not disappear until either immediately before or sometime after the sieve plates are fully perforated, for nuclei were found in sieve elements at all stages of sieve-plate perforation in all 13 species. Considerable variation also existed in the morphology of nuclei encountered in mature sieve-tube members (Table). Most nuclei appeared more or less degenerated, either as clear spherical bodies (Figs. 13, 15-17, 23, 24, 31), sometimes swollen (Figs. 18, 22), or as small dense bodies (Figs. 14, 19), with intermediate forms (Figs. 20, 25). Many of the clear nuclei contained a single, nucleolus-like structure (Figs. 15-17, 27, 28, 32-34). Nuclei of normal appearance were found in mature sieve elements
292
~. F. Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 26--34
Nuclei in Mature Sieve Elements
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of only three of the woody dicotyledons: Robinia (compare nucleus of immature element, Fig. 26, with those of mature elements, Figs. 27, 28), Ulmus and Vitis (Figs. 33, 34). Many nuclei occurred near sieve plates. Some were found within the shme plugs, and were barely visible (Fig. 23). Mature sieve-tube members with nuclei were distributed throughout the growth increments and were found even in some elements that had recently become nonfunctional and from which most cytoplasmic contents had disappeared, as in the Aeer saceharinum sieve element of Fig. 16 (see also the Ulmus sieve element of Fig. 76, Evert et al., 1969). Several second-year (reactivated) sieve elements of Tilia (Figs. 29, 30) and Vitis also contained nuclei. The sieve elements of four of the woody dicotyledons (Populus, Quereus, Salix and Tilia) contained spherical inclusions similar to those commonly interpreted as extruded nueleoli, but which recently have been reported not to be nueleoli (Deshpande and Evert, 1970). One of these spherical inclusions in an immature Populus sieve element is shown in Fig. 21. Another (somewhat out of the plane of focus) can be seen in the mature Populu8 sieve element of Fig. 22. Of the tissues processed according to the Feulgen technique, nuclei were detected in mature sieve elements of only Robinia and Ulmus. The results for Robinia were essentially similar to those reported elsewhere for Ulmus (Evert et al., 1969); that is, the nuclei of most mature sieve elements gave a light staining reaction similar to that of nuclei in immature sieve elements and adjaeent parenchyma cells. Dense, spherical bodies similar to those identified as nuclei in other preparations of Robinia tissue gave very dense staining reactions. Discussion
As mentioned, one of the goals of the present investigation was to determine the stage of development that nuclear degeneration occurs in sieve cells of Metasequoia. Although Kollmann (1964) reported that a nucleus "is seen in sieve cells near the cambium with well developed plasmic connections", no indication was given of the longevity of the nuclei. The present study indicates that nuclear disintegration does not occur until the pertinent sieve elements begin to degenerate or to cease functioning. Apparently, this is also true of the sieve elements in Sequoia
Figs. 26-34. Longitudinal (Figs. 26 and 27, 29-34) and transverse (Fig. 28) views of mature (Figs. 27 34) and immature (Fig. 26) sieve.tube members with nuclei. Figs. 26~8 : Robinia pseudoacacia. • 640, • 400, • 400, respectively. Figs. 29, 30 : Tilia americana. Fig. 29 shows p~rt of sieve-tube member of Fig. 30 a~ higher magnification. X 1000 and X400. Fig. 31: Ulmus americana. X470. Figs. 32-34: Vitis riparia. Fig. 34 shows part of sieve-tube member of Fig. 33 at higher magnification. • 640, • 275, • 640, respectively. B slime body; PL slime plug; SP sieve plate
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and Taxodium. The presence of nuclei of normal appearance or morphology in all functional sieve cells of Metasequoia, Sequoia and Taxodium constituted the most surprising result of the present investigation. The occurrence of nuclei in mature sieve-tube members of 12 of the 13 species of woody dicotyledons examined during the present study substantially increases the list of angiosperms containing some mature sieve elements with persistent nuclei. As mentioned, in all 13 species nuclei were found in sieve elements, at all stages of sieve-plate perforation. Only Salix nigra lacked nuclei in fully-perforated sieve elements beyond the immediate vicinity of the cambium. On the other hand, in the other 12 species, nuclei were found in mature sieve elements of all ages, including some second-year elements of Tilia and Vitis, dicotyledons long known to contain sieve elements t h a t undergo reactivation in spring and function for parts of two or more growing seasons (Esau, 1948, 1965; I-Ioldheide, 1951; Evert, 1962; Davis and Evert, 1970). Of the 12 dicotyledons containing some mature sieve elements with persistent nuclei, fewer nucleate elements were encountered in Tilia than in any other. Earlier, both tubular and fibrillar forms of slime or P protein were found in nuclei of differentiating sieve elements of Tilia with the electron microscope (Evert and Deshpande, 1970). No nuclei were found in mature Tilia sieve elements at that time. There can be no doubt t h a t the sieve cells of the secondary phloem of Metasequoia, Sequoia and Taxodium contain nuclei of normal appearante at a time when they are involved with long-distance assimilate transport, for the sieve cells in these conifers retain such nuclei until they begin to form definitive callose. The situation in the woody dicotyledons is not so dear-cut. Only Robinia, Ulmus and Vitis contained apparently functional sieve-tube members with nuclei of normal appearance. None of the numerous nuclei encountered in mature sieve-tube members of the two Cornus species was normal in appearance. Although the great mass of data accumulated over the years with both light and electron microscopes (Esau, 1969, p. 65) points to a sieve element that is enucleate at maturity or at a time when it is involved with the long-distance transport of assimilates, the possibility exists t h a t m a n y of the morphological changes of nuclei associated with sieve-element differentiation and the disappearance of the nucleus from the sieve element are induced during manipulation and fixation of the tissue. I t long has been recognized t h a t the sieve-element protoplast becomes increasingly sensitive to manipulation and fixation as it approaches maturity and that at maturity is extremely labile (Esau, 1969, p. 80). Experimental studies are currently in progress in an effort to resolve this important problem of the precence of nuclei in functional sieve elements.
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References Alfieri, F. J., Evert, R. F. : Seasonal development of the secondary phloem in Pinus. Amer. J. Bot. 55, 518-528 (1968). Cheadle, V. L, Gilford, E. M., Jr., Esau, K. : A staining combination for phloem and contiguous tissues. Stain Technol. 28, 49-53 (1953). Davis, J. D., Evert, R. F. : Seasonal cycle of phloem development in woody vines. Bot. Gaz., in press (1970). Deshpande, B. P., Evert, R. F. : A reevaluation of extruded nucleoli in sieve elements. J. Ultrastruct. Res., in press (1970). Ervin, E. L., Evert, R. F. : Observations on sieve elements in three perennial monocotyledons. Amer. J. Bot. 57, 218-225 (1970). Esau, K. : Phloem structure in the grapevine, and its seasonal changes. Hilgardia 18, 217-296 (1948). - - Anatomy and cytology of Vitis phloem. Hilgardia 37, 17-72 (1965). - - The phloem. In: Handbuch der Pflanzenanatomie, W. Zimmerman, P. 0zenda, H. D. Wulff, ed., vol. 5, pt. 2. Berlin-Stuttgart: Borntraeger 1969. Evert, R . F . : Some aspects of phloem development in Tilia americana. (Abstr.) Amer. J. Bot. 49, 659 (1962). - - Alfieri, F. J. : Ontogeny and structure of coniferous sieve cells. Amer. J. Bot. 52, i058-1066 (1965). - - Deshpande, B. P. : Nuclear P protein in sieve elements of Tilia americana. J. Cell Biol. 44, 462-466 (1970). - - Tucker, C. M., Davis, J. D., Deshpande, B. P. :Light microscope investigation of sieve-element ontogeny and structure in Ulmus americana. Amcr. J. Bot. 56, 999-1017 (1969). Fischer, A. : Neue Beitr~ge zur Kenntnis der SiebrShren. Ber. Verh. K5n. Siiehs. Ges. Wiss. Leipzig, Math.-Phys. C1.38, 291-336 (1886). Holdheide, W. : Anatomic mitteleuropi~ischer Geh51zrinden. In: H. Freund's Handbuch der Mikroskopie in der Technik, vol. 5, pt. 1, p. 193-367. Frankfurt a. Main: Umschau 1951. Kollmann, R. : On the fine structure of the sieve element protoplast. Phytomorph. 14, 247-264 (1964). - - Schumacher, W.: Uber die Feinstruktur des Phloems yon Metasequoia glyptostroboides und seine jahreszeitlichen Ver~nderungen. V. Die Differenzierung der Siebzellen im Verlaufe einer Vegetationsperiode. Planta (Berl.) 63, 155-190 (1964). Lecomte, H.: Contribution s l'6tude du liber des Angiospermes. Ann. Sci. Nat., S@r. VII, Bot. 10, 193-324 (1889). Murmanis, L., Evert, R . F . : Some aspects of sieve cell ultrastructure in Pinus strobus. Amer. J. Bot. 53, 1065-1078 (1966). O'Brien, T. P., Thimann, K. V. : Observations on the fine structure of the oat coleoptile. III. Correlated light and electron microscopy of the vascular tissues. Protoplasma (Wien) 63, 443-478 (1967). Paolillo, D. J., Jr.: The developmental anatomy of Isoetes. Illinois Biol. Monogr. No. 31. Urbana: Illinois Univ. Press 1963. Parthasarathy, M. V. : Studies on metaphloem in petioles and roots of Palmae. Doer. Diss., Cornell University, Ithaca, N.Y. 1966. Sass, J . E . : Botanical microtechnique, 3d ed., Ames: Iowa State Coll. Press 1958. Schmidt, E . W . : Bau und Funktion der Siebr6hre der Angiospermen. Jena: Fischer I917.
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Scott, D. H., Brebner, G. : On the anatomy and histogeny of Strychnos. Ann. Bot. 3 , 275-304 (1889). Shah, J. J., James, M. R. : Sieve tube elements in the stem of 3/el)tunia oleracea Lour. Aust. J. Bot. 16, 433444 (1968). Srivastava, L. M. : The secondary phloem of Austrobaileya scandens. Canad. J. Bot. 48, 341-359 (1970). -O'Brien, T. P. : On the ultrastrueture of cambium and its vascular derivatives. II. Secondary phloem of Pinus strobus. Protoplasma (Wien) 61, 277-293 (1966). Tucker, C. M., Evert, R. F. : Seasonal development of the secondary phloem in Acer negundo. Amer. J. Bot. 56, 275-284 (1969). Wooding, F. B. P. : The development of the sieve elements of Pinus pinea. Planta (Berl.) 69, 230-243 (1966). Prof. R. F. Evert Department of Botany University of Wisconsin Madison, Wisconsin 53706, U.S.A.
C. Milton Tueker's present address: Department of Natural Sciences Freed-Hardeman College Henderson, Tennessee 38340, U.S.A.
Jerry D. Davis' present address: Department of Biology Wisconsin State University LaCrosse, Wisconsin 54601, U.S.A.
Frank J. Alfieri's present address: Department of Biology California State College Long Beach, California 90804, U.S.A.
On the Occurrence of Nuclei in Mature Sieve Elements* RAY F. EV~RT, JEI~RY D. DAvls, C. MILTON Tucl;~a and FRANK J. ALFIERI Department of Botany, University of Wisconsin, Madison l~eceived July 23 / September 18, 1970 Summary. The secondary phloem of 3 species of the Taxodiaccae and 13 species of woody dicotyledons was examined for the occurrence of nuclei in mature sieve elements. Nuclei were found in all mature sieve cells of Metasequoia qlyptostroboides, Sequoia sempervirens and Taxodium distiehum, and in some mature sieve-tube members in 12 of the 13 species of woody dicotyledons. Except for nuclei of sieve cells undergoing cessation of function, the nuclei in mature sieve cells of M. glyptostroboides, S. sempervirens and T. distiehum were normal in appearance. The occurrence and morphology of nuclei in mature sieve-tube members of the woody dicotyledons were quite variable. Only 3 species, Robinia pseudoaeacia, Ulmus americana and Vitis riparia, contained some mature sieve elements with apparently normal nuclei. Introduction P h l o e m i n v e s t i g a t o r s g e n e r a l l y agree t h a t t h e sieve e l e m e n t lacks a nucleus a t m a t u r i t y . E a r l y accounts of t h e presence of nuclei in m a t u r e sieve elements (Fischer, 1886 ; Lecomte, 1889 ; S c o t t a n d Brebner, 1889 ; S c h m i d t , 1917) were g e n e r a l l y dismissed as m i s i n t e r p r e t a t i o n s . H o w e v e r , during t h e p a s t few y e a r s new r e p o r t s h a v e been m a d e of t h e presence of nuclei in some m a t u r e sieve elements in a wide v a r i e t y of v a s c u l a r p l a n t s , including Isoetes (Paolillo, 1963), several species of conifers ( E v e r t a n d Alfieri, 1965; M u r m a n i s a n d E v e r t , 1966; S r i v a s t a v a a n d O'Brien, 1966; W o o d i n g , 1966), Austrobaileya ( S r i v a s t a v a , 1970), Neptunia (Shah a n d J a m e s , 1968), Ulmus ( E v e r t et al., 1969), Secale ( 0 ' B r i e n a n d Thim a n n , 1967), Smilax (Ervin a n d E v e r t , 1970) a n d some of t h e p a l m s ( P a r t h a s a r t h y , 1966). I n m o s t cases these nuclei were described as being d e g e n e r a t e d or necrotic in a p p e a r a n c e . Nuclei of n o r m a l a p p e a r a n c e or m o r p h o l o g y were e n c o u n t e r e d in m a t u r e sieve elements in o n l y two of t h e species i n v e s t i g a t e d , Smilax hispida (Ervin a n d E v e r t , 1970) a n d Ulmus americana ( E v e r t et al., 1969). N o n e of the r e c e n t i n v e s t i g a t i o n s cited a b o v e was u n d e r t a k e n p r i m a r i l y to d e t e r m i n e t h e occurrence of nuclei in m a t u r e sieve elements. Because * This research has been supported by National Science Foundation grants GB-5950 and GB-8330. 19 Planta (Berl.), Bd. 95
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Table. Occurrence and morphology o] nuclei in mature sieve elements Species
Occurrence
Morphology
Metasequoia glyptostroboides Hu and Cheng Sequoia sempervirens (D. Don) Endl.
In all elements In all elements In all elements Occasional
Normal
Taxodium distichum (L.) Rich. Acer negundo L. Acer saccharinum L. Comus racemosa Lam. Comus stoloui/era Miehx. Juglans nigra L.
Occasional Frequent Occasional to frequent Occasional
Populus tremuloides Michx.
Occasional
Quercus alba L. Rhus glabra L.
Occasional Occasional
Robinia pseudoacacia L.
Frequent
Salix nigra Marsh. Tilia americana L. Ulmus americana L.
None Occasional Occasional to frequent
Vitis riparia Michx.
Occasional
Normal Normal Clear to dense spherical body Clear spherical body Clear spherical body Dense spherical to elongate body Relatively dense spherical body Relatively clear spherical body, sometimes swollen Clear spherical body Clear to dense spherical body Normal to dense spherical body Clear crumpled body Normal to clear spherical body, sometimes swollen Normal to clear spherical body
of the recent reports of nuclei in mature sieve elements and of the importance of this feature in the overall understanding of sieve-element ontogeny, structure and function, the present investigation was undertaken. The species of woody dicotyledons selected for investigation (Table) were largely those already chosen for studies of seasonal phloem development and for which extensive, year-round collections of tissues were available. Three conifers, Metasequoia gIyptostroboides, Sequoia sempervirens and Taxodium distichum, all members of the Taxodiaceae, were also examined.
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Metasequoia was of special interest, for its sieve cells have been studied intensively a t the ultrastructural level b y Kollmann a n d Schumacher (1964, and literature cited therein). I n a review article, Kollmann (1964) reported t h a t a n u c l e u s - - " t h o u g h of changed shape " - - c o u l d still be found in sieve cells with well developed connections. Although he reported briefly on the process of nuclear disintegration in Metasequoia sieve cells, he did not state at what age or stage of sieve cell developm e n t t h a t process took place. One of the goals of the present investigation was to answer t h a t question.
Materials and Methods The Metasequoia glypto~troboides material was obtained from 2-to-5-year-old shoots of trees grown in the greenhouse and out-of-doors in Madison, Wisconsin. Two to four trees were sampled on each of 16 collection dates over a 3-yearperiod. Tissues were collected on the following dates: 29 J a n u a r y 1966, 2 F e b r u a r y 1967, 14 and 20 March 1966, 9 a n d 20 April 1965, 8 and 20 April 1966, 5 April 1967, 29 May 1966, 18 a n d 24 J u n e 1965, 17 J u n e 1966, 1 J u l y 1966, and 5 and 29 November 1966. The Sequoia sempervirens material was collected on 15 November 1967, through the courtesy of the Crown Simpson Corporation, San Francisco, California. The Taxodium distichum material was collected on 28 August 1967, at Henderson, Tennessee. Tissues of b o t h were obtained from the trunks of apparently healthy trees. Most tissues were immersed immediately in formaldehyde-acetic acid-alcohol (FAA; Sass, 1958, p. 15) and shortly afterward aspirated in the laboratory. A]] tissues were embedded in celloidin and sectioned with a sliding microtome a t 15 ~z. Serial transverse, radial and tangential sections were obtained of all collections. The sections were tied onto slides with thread, stained with tannic acid-ferric chloride-resorcin blue (Cheadle et al., 1953), and then mounted in Harleco synthetic resin. Seasonal collections were available of all woody dicotyledons (Table) except Robinia. Tissues were obtained from the trunks of apparently healthy trees 7 or more years old growing in the Eagle Heights area of the University of Wisconsin campus and in the University of Wisconsin Arboretum at Madison. Two to four trees were sampled a t each collection date. The n u m b e r of collection dates ranged from 14 over a I-year-period for Cornus racemosa, Juglans nigra, Salix nigra and Ulmus americana to 64 over a 2-year-period for Acer negundo, Rhus glabra and Tilia americana. All months of the year were represented. Collection procedures were the same as those outlined by Tucker and Everg (1969) for a study of seasonal phloem development in Acer negundo. The materials were placed in either chromic acid-acetic aeid-formMdehyde (Craf I I I ; Sass, 1958, p. 18) or F A A in the field a n d aspirated shortly afterward in the laboratory. Serial transverse and radial sections were obtained of all collections, and serial tangential sections of selected ones. As with the conifer sections, these were stained by the method of Cheadle et al. (1953). The Vitis tissues were the same as those used in a recent study of seasonal phloem development in vines (Davis and Evert, 1979). I n addition to the seasonal collections, other tissues of the dicotyledonous species (with the exception of Juglans nigra, Rhu8 glabra and Viti8 r@aria) were collected during the summer of 1967, on the following dates: 8 and 19 June, 10 July, 9 August, a n d 18 September. All these tissues, including those of Robinia, were treated in the variety of ways outlined for the tissues of Ulmus collected on the same dates in 1967, as reported in a recent article (Evert et al., 1969). I n addition, as with the Ulmus study, examination of fixed materials was supplemented with t h a t of fresh material. 19"
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t~. F. Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 1--4
Nuclei in Mature Sieve Elements
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Results Taxodiaceae. The s e c o n d a r y p h l o e m of Metasequoia (Figs. 1-6), Sequoia (Figs. 7-9) a n d Taxodium (Figs. 10-12) is v e r y similar in composition a n d a r r a n g e m e n t . The axial s y s t e m consists of fibers, sieve cells, s t r a n d s of p a r e n c h y m a cells, a n d a l b u m i n o u s cells. The m o s t l y uniseriate r a y s consist o5 o r d i n a r y r a y p a r e n c h y m a cells a n d a l b u m i n o u s cells, t h e l a t t e r g e n e r a l l y occurring on t h e m a r g i n s of t h e rays. Cells of t h e axial s y s t e m a l t e r n a t e r a d i a l l y in a sequence of sieve cells, p a r e n c h y m a cells, sieve cells, fibers, sieve cells, p a r e n c h y m a cells, a n d so on (Figs. 1, 2, 5, 6, 10, 11). Local irregularities do occur, however, a n d are m o s t c o m m o n n e a r t h e ends of t h e g r o w t h increments. The fibers of the e a r l y p h l o e m are c o m m o n l y wider a n d h a v e t h i c k e r walls t h a n those of the ]ate p h l o e m ; therefore, g r o w t h i n c r e m e n t s generally are easily d e l i m i t e d in all t h r e e species. This f e a t u r e was of g r e a t value in t h e i n t e r p r e t a t i o n of t h e sequence of events in t h e d e v e l o p m e n t of the s e c o n d a r y p h l o e m of Metasequoia a n d in t h e d e t e r m i n a t i o n of t h e a p p r o x i m a t e ages of t h e sieve cells in all t h r e e species. Mature sieve cells were identified through the presence of fully-developed sieve areas and of crystals in the radial walls. As in Pinus (Evert and Alfieri, 1965), the sieve-area pores in Metasequoia, Sequoia and Taxodium occur in groups. AIthough individual pores are difficult to detect in living sieve cells with the light microscope, fully-developed pore groups are detectable, if only with aid of an oilimmersion objective. Unlike their counterparts in the Pinaceae, mature sieve cells in the Taxodiaceae lack secondary walls. All mature cell types in the secondary phloem of the species of Taxodiaeeae examined during the present study are characterized by the presence of numerous, small crystals in the middle lamella of the radial walls, a phenomenon reported earlier for the secondary phloem of Juniperus (Evert and Alfieri, 1965). The crystals, which are absent at the sieve areas and primary pit-fields, are lacking in cambial zone cells and in immature ceils adjacent to the cambium. Although the present article is not concerned with the seasonal cycles of phloem development of the species examined, a thorough understanding of the nuclear condition of the sieve cells in Metasequoia is virtually impossible without certain aspects of its seasonal cycle of phloem development first being understood. The seasonal cycle of phloem development of Metasequoia is, in some respects, similar to that of Pinus (Alfieri and Evert, 1968). In both, all hut the last-formed sieve
Figs. 1-4. Metasequoia glyptostroboides. Fig. 1. Transection showing some xylem, the dormant cambial zone, and mostly nonfunctional phloem. Unlabeled arrows point to nuclei in mature sieve cells. Collected 5 November 1966. • 610. Fig. 2. Radial section showing some xylem, the dormant cambial zone, some functional and nonfunctional phloem. Unlabeled arrows point to sieve areas containing variable amounts of eallose. Collected 5 November 1966. • Figs. 3, 4. Radial and tangential sections, respectively, showing nuclei (unlabeled arrows) in mature, overwintering sieve cells. Collected 5 April 1967. • CZ cambial zone; F fiber; P parenchyma cell; /~ ray; S sieve cell; X xylem
286
1~. F. Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 5--9
Nuclei in Mature Sieve Elements
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cells cease functioning the same year they are derived from the cambium. The former overwinter and most remain functional until new sieve ceils differentiate in spring. The first new sieve cells of spring develop directly from cells which overwintered in an undifferentiated state on the outer margin of the cambial zone. At about the same time, or shortly afterward, eambial activity is resumed and new phloem initials are produced. In Pinus, the sieve cells that overwinter in a mature state generally do not become nonfunctional until after early phloem differentiation is completed in late May or June in the Madison, Wisconsin area. In Mstasequoia, cessation of function of the overwintering sieve cells occurs much earlier, being initiated in some cells before, and in others shortly after the initiation of new sieve cell differentiation in April. Figs. 1 a n d 2 i l l u s t r a t e t h e condition of the c a m b i a l zone a n d t h e c u r r e n t y e a r ' s p h l o e m i n c r e m e n t in a N o v e m b e r collection of Metasequoia. E x c e p t for two to t h r e e layers of sieve cells n e a r e s t t h e c a m b i u m , t h e sieve cells c o n t a i n e d conspicuous a m o u n t s of definitive eallose (Fig. 2). The sieve cells f a r t h e s t from t h e c a m b i u m - - t h o s e containing the g r e a t e s t a m o u n t s of c a l l o s e - - w e r e a l r e a d y nonfunctional. Those n e a r e s t t h e cambium, t h e last two to t h r e e layers which c o m m o n l y overwinter in a f u n c t i o n a l state, c o n t a i n e d p r o t o p l a s t s w i t h nuclei of n o r m a l a p p e a r a n c e (Fig. 1): The p r o t o p l a s t s of sieve cells between these two e x t r e m e s were in v a r y i n g stages of d e g e n e r a t i o n a n d c o n t a i n e d nuclei more or less necrotic in a p p e a r a n c e . I n all w i n t e r collections t h e two to three layers of overwintering, m a t u r e sieve cells c o n t a i n e d nuclei which were n o r m a l in a p p e a r a n c e . I n a d d i t i o n , their c o u n t e r p a r t s in t h e A p r i l collections also c o n t a i n e d such nuclei (Figs. 3, 4), e x c e p t in cells undergoing cessation of function a t t h a t time. Fig. 5 is a view of the c a m b i a l region a n d a d j a c e n t s e c o n d a r y p h l o e m of a n o t h e r A p r i l collection. H e r e t h e o u t e r m o s t l a y e r of m a t u r e sieve cells c o n t a i n e d s u b s t a n t i a l a m o u n t s of definitive callose, while t h e l a y e r n e a r e s t t h e c a m b i u m h a d v e r y little. The u n l a b e l e d arrow p o i n t s to a l a y e r of u n d i f f e r e n t i a t e d cells which, j u d g i n g from other collections, n o r m a l l y would h a v e d e v e l o p e d d i r e c t l y into t h e first, new sieve cells of spring.
Figs. 5 and 6. Metasequoia glyptostroboides. Fig. 5. Transection showing some xylem, the dormant cambial zone, two bands of mature, overwintering sieve cells, and nonfunctional phloem. Definitive callose (C) has begun to accumulate in outermost band of overwintering sieve cells. Unlabeled arrow points to layer of probable sieve-cell primordia. Collected 20 April 1966. • 400. Fig. 6. Radial section showing nuclei (unlabeled arrows) in mature sieve cells. Collected 24 June 1965. X400 Figs. 7-9. Sequoia sempervirens. Fig. 7. t~.adial section showing nuclei in mature sieve cells farthest from the cambium. Figs. 8, 9 (radial and tangential sections, respectively). Normal-appearing nuclei (unlabeled arrows) in mature sieve cells. X440. C callose; CZ cambial zone; F fiber; P parenchyma cell; S sieve cell
288
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Figs. 10-12. Taxodium distichum. Fig. 10. Transection showing cambial zone and mostly functional phloem. Unlabeled arrow points to nucleus in mature sieve cell. X250. Figs. 11, 12. Radial sections showing nuclei (unlabeled arrows) in mature sieve cells. The sieve cell in Fig. 12 was in band of sieve cells farthest from the cambium. Its nucleus is necrotic in appearance, x 400. CZ cambial zone; F fiber; P parenchyma cells; R ray; S sieve cell; S A sieve area
Figs. 13-19. Views of mature sieve-tube members with nuclei (unlabeled arrows) in longitudinal (Figs. 13-16, 18 and 19) and transverse (Fig. 17) sections. The nucleate sieve element in Fig. 16 is nonfunctional. Figs. 13, 14: Acer negundo. • 640. Figs. 15, 16: Acer saccharinum, x400 and • 640, respectively. Figs. 17, 18: Comus racemosa. • Fig. 19: Comus stoloni/era. • CZ cambia] zone; P parenchyma cell; S sieve-tube member; S P sieve plate; X xylem
Nuclei in M~turc Sieve Elements
Figs. 13--19
289
290
R. F, Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 20-25. Longitudinal views of mature (Figs. 20, 22-25) and immature (Fig. 21) sieve-tube members with nuclei (unlabeled arrows) Fig. 20: Juglans nigra. • 640. Figs. 21, 22: Populus tremuloides. Arrow heads point to sphericalinclusions. • 640. Fig. 23. Quercus alba. • 640. Figs. 24, 25: Rhus glabra. • 640 and • 500, respectively. S P sieve plate
Nuclei in Mature Sieve Elements
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Subsequent seasonal collections showed t h a t the retention of nuclei of normal appearance until the cell begins to cease functioning, determined by the accumulation of definitive callose at the sieve areas and by degeneration of the protoplast, is not unique to overwintering sieve cells, but is common for all sieve cells in the secondary phloem of Metasequoia. Fig. 6 is a radial view of a newly-formed, although incomplete, phloem increment of a late June collection. Nuclei (arrows) can be seen in mature cells of the fourth, fifth and sixth layers of sieve cells from the cambium. These are representative of all mature, presumably functional sieve cells in the secondary phloem of Metasequoia. The Feulgen reaction was performed on several collections of Metasequoia tissue and, in each, the nuclei of all cell t y p e s - - b o t h mature and immature sieve cells, parenchyma cells, albuminous cells, and cambial cells--gave strongly positive staining reactions. All of the mature sieve cells encountered in the secondary phloem of Sequoia and Taxodium contained nuclei. In both collections only the outermost one or two layers of sieve cells--sieve cells undergoing cessation of function--contained nuclei more or less necrotic in appearance. In Sequoia the nuclei in such cells had a dense, almost amorphous appearance (Fig. 7) while in comparable cells of Taxodium the nuclear envelope appeared to be separating from the rest of the nucleus (Fig. 12). Figs. 8 to l l illustrate nuclei representative of those found in the majority of mature sieve cells in Sequoia and Taxodium. Woody Dicotyledons. The occurrence of nuclei in mature sieve-tube members (i. e., in sieve-tube members with fully-perforated sieve plates) of the 13 species of woody dicotyledons was quite variable (Table). However, nuclei were encountered in some mature sieve elements in all but one of them, namely, Salix nigra. Nuclei occurred occasionally in mature sieve elements of eight species and frequently in two others. Within still two other species, Comus stoloni/era and Ulmus americana, the occurrence of nuclei ranged from occasional in some collections to frequent in others. If nuclei are normally absent from mature sieve elements of any of the species examined, they apparently do not disappear until either immediately before or sometime after the sieve plates are fully perforated, for nuclei were found in sieve elements at all stages of sieve-plate perforation in all 13 species. Considerable variation also existed in the morphology of nuclei encountered in mature sieve-tube members (Table). Most nuclei appeared more or less degenerated, either as clear spherical bodies (Figs. 13, 15-17, 23, 24, 31), sometimes swollen (Figs. 18, 22), or as small dense bodies (Figs. 14, 19), with intermediate forms (Figs. 20, 25). Many of the clear nuclei contained a single, nucleolus-like structure (Figs. 15-17, 27, 28, 32-34). Nuclei of normal appearance were found in mature sieve elements
292
~. F. Evert, J. D. Davis, C. M. Tucker and F. J. Alfieri:
Figs. 26--34
Nuclei in Mature Sieve Elements
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of only three of the woody dicotyledons: Robinia (compare nucleus of immature element, Fig. 26, with those of mature elements, Figs. 27, 28), Ulmus and Vitis (Figs. 33, 34). Many nuclei occurred near sieve plates. Some were found within the shme plugs, and were barely visible (Fig. 23). Mature sieve-tube members with nuclei were distributed throughout the growth increments and were found even in some elements that had recently become nonfunctional and from which most cytoplasmic contents had disappeared, as in the Aeer saceharinum sieve element of Fig. 16 (see also the Ulmus sieve element of Fig. 76, Evert et al., 1969). Several second-year (reactivated) sieve elements of Tilia (Figs. 29, 30) and Vitis also contained nuclei. The sieve elements of four of the woody dicotyledons (Populus, Quereus, Salix and Tilia) contained spherical inclusions similar to those commonly interpreted as extruded nueleoli, but which recently have been reported not to be nueleoli (Deshpande and Evert, 1970). One of these spherical inclusions in an immature Populus sieve element is shown in Fig. 21. Another (somewhat out of the plane of focus) can be seen in the mature Populu8 sieve element of Fig. 22. Of the tissues processed according to the Feulgen technique, nuclei were detected in mature sieve elements of only Robinia and Ulmus. The results for Robinia were essentially similar to those reported elsewhere for Ulmus (Evert et al., 1969); that is, the nuclei of most mature sieve elements gave a light staining reaction similar to that of nuclei in immature sieve elements and adjaeent parenchyma cells. Dense, spherical bodies similar to those identified as nuclei in other preparations of Robinia tissue gave very dense staining reactions. Discussion
As mentioned, one of the goals of the present investigation was to determine the stage of development that nuclear degeneration occurs in sieve cells of Metasequoia. Although Kollmann (1964) reported that a nucleus "is seen in sieve cells near the cambium with well developed plasmic connections", no indication was given of the longevity of the nuclei. The present study indicates that nuclear disintegration does not occur until the pertinent sieve elements begin to degenerate or to cease functioning. Apparently, this is also true of the sieve elements in Sequoia
Figs. 26-34. Longitudinal (Figs. 26 and 27, 29-34) and transverse (Fig. 28) views of mature (Figs. 27 34) and immature (Fig. 26) sieve.tube members with nuclei. Figs. 26~8 : Robinia pseudoacacia. • 640, • 400, • 400, respectively. Figs. 29, 30 : Tilia americana. Fig. 29 shows p~rt of sieve-tube member of Fig. 30 a~ higher magnification. X 1000 and X400. Fig. 31: Ulmus americana. X470. Figs. 32-34: Vitis riparia. Fig. 34 shows part of sieve-tube member of Fig. 33 at higher magnification. • 640, • 275, • 640, respectively. B slime body; PL slime plug; SP sieve plate
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and Taxodium. The presence of nuclei of normal appearance or morphology in all functional sieve cells of Metasequoia, Sequoia and Taxodium constituted the most surprising result of the present investigation. The occurrence of nuclei in mature sieve-tube members of 12 of the 13 species of woody dicotyledons examined during the present study substantially increases the list of angiosperms containing some mature sieve elements with persistent nuclei. As mentioned, in all 13 species nuclei were found in sieve elements, at all stages of sieve-plate perforation. Only Salix nigra lacked nuclei in fully-perforated sieve elements beyond the immediate vicinity of the cambium. On the other hand, in the other 12 species, nuclei were found in mature sieve elements of all ages, including some second-year elements of Tilia and Vitis, dicotyledons long known to contain sieve elements t h a t undergo reactivation in spring and function for parts of two or more growing seasons (Esau, 1948, 1965; I-Ioldheide, 1951; Evert, 1962; Davis and Evert, 1970). Of the 12 dicotyledons containing some mature sieve elements with persistent nuclei, fewer nucleate elements were encountered in Tilia than in any other. Earlier, both tubular and fibrillar forms of slime or P protein were found in nuclei of differentiating sieve elements of Tilia with the electron microscope (Evert and Deshpande, 1970). No nuclei were found in mature Tilia sieve elements at that time. There can be no doubt t h a t the sieve cells of the secondary phloem of Metasequoia, Sequoia and Taxodium contain nuclei of normal appearante at a time when they are involved with long-distance assimilate transport, for the sieve cells in these conifers retain such nuclei until they begin to form definitive callose. The situation in the woody dicotyledons is not so dear-cut. Only Robinia, Ulmus and Vitis contained apparently functional sieve-tube members with nuclei of normal appearance. None of the numerous nuclei encountered in mature sieve-tube members of the two Cornus species was normal in appearance. Although the great mass of data accumulated over the years with both light and electron microscopes (Esau, 1969, p. 65) points to a sieve element that is enucleate at maturity or at a time when it is involved with the long-distance transport of assimilates, the possibility exists t h a t m a n y of the morphological changes of nuclei associated with sieve-element differentiation and the disappearance of the nucleus from the sieve element are induced during manipulation and fixation of the tissue. I t long has been recognized t h a t the sieve-element protoplast becomes increasingly sensitive to manipulation and fixation as it approaches maturity and that at maturity is extremely labile (Esau, 1969, p. 80). Experimental studies are currently in progress in an effort to resolve this important problem of the precence of nuclei in functional sieve elements.
Nuclei in Mature Sieve Elements
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Scott, D. H., Brebner, G. : On the anatomy and histogeny of Strychnos. Ann. Bot. 3 , 275-304 (1889). Shah, J. J., James, M. R. : Sieve tube elements in the stem of 3/el)tunia oleracea Lour. Aust. J. Bot. 16, 433444 (1968). Srivastava, L. M. : The secondary phloem of Austrobaileya scandens. Canad. J. Bot. 48, 341-359 (1970). -O'Brien, T. P. : On the ultrastrueture of cambium and its vascular derivatives. II. Secondary phloem of Pinus strobus. Protoplasma (Wien) 61, 277-293 (1966). Tucker, C. M., Evert, R. F. : Seasonal development of the secondary phloem in Acer negundo. Amer. J. Bot. 56, 275-284 (1969). Wooding, F. B. P. : The development of the sieve elements of Pinus pinea. Planta (Berl.) 69, 230-243 (1966). Prof. R. F. Evert Department of Botany University of Wisconsin Madison, Wisconsin 53706, U.S.A.
C. Milton Tueker's present address: Department of Natural Sciences Freed-Hardeman College Henderson, Tennessee 38340, U.S.A.
Jerry D. Davis' present address: Department of Biology Wisconsin State University LaCrosse, Wisconsin 54601, U.S.A.
Frank J. Alfieri's present address: Department of Biology California State College Long Beach, California 90804, U.S.A.
