ULTRASTRUCTURE OF DEVELOPING SIEVE ELEMENTS IN THLASPI ARVENSE L. II. MATURATION

Developing sieve elements of pennycress (Thlaspi arvense L.) were studied with the electron microscope. The maturation of sieve elements involved loss of ribosomes from cytoplasm; degeneration of nulcei; modification of endoplasmic reticulum (ER); loss of tonoplast; and disappearance of dictyosomes and dictyosomes vesicles, coated vesicles, microtubules, and microbodies. Such changes produce a mature, presumably conducting cell that contains no nucleus or central vacuole but which retains a thin layer of peripheral cytoplasm with plastids, mitochondria, and smooth ER. Some similar changes have been described in a variety of developing sieve elements of angiosperms, but coated vesicles and microbodies previously have not been followed through sieve-element maturation. Likewise, few developmental studies have been made of plant sieve elements that exhibit two types of P-protein, the tubular type and the granular P-protein body.     

P-protein and inclusion bodies in root protophloem of Salix viminalis

The formation of P-protein in the protophloem of 9- to 14-day-old adventitious roots of Salix viminalis was studied. In immature sieve elements a finely granular material was present. This was considered to be nascent P-protein. Small aggregations of tubular P-protein were observed 17 cells from the first "cleared" sieve element. In older cells the bodies were up to 7 μm long. Nondispersed and disaggregating P-protein bodies were present in mature sieve elements. P-protein bodies were also observed in parenchyma cells adjoining mature sieve elements. In addition, inclusion bodies of unknown origin are described. They had a granular content and were most often found in mature sieve elements.     

Electron microscope investigation of sieve-element ontogeny and structure inUlmus americana

Summary During advanced stages of sieve-element differentiation inUlmus americana L., dispersal of the P-protein (slime) bodies results in formation of a peripheral network of strands consisting of aggregates of P-protein components having a striated, fibrillar appearance. The tonoplast is present throughout the period of P-protein body dispersal. Perforation of the sieve plates is initiated during early stages of P-protein body dispersal.Small P-protein bodies consist of tubular components, most of which measure about 180 Å in diameter. With increase in size of the P-protein bodies narrower components appear. At the time of initiation of P-protein body dispersal, most of the components comprising the bodies are of relatively narrow diameters (most 130–140 Å) and have a striated, fibrillar appearance. Both wide and narrow P-protein components are present throughout the period of sieve-element differentiation and in the mature cell as well, and a complete intergradation in size and appearance exists between the two extremes. Both extremes of P-protein component have a similar substructure: an electron-transparent lumen and an electronopaque wall composed of subunits, apparently in helical arrangement. The distribution of P protein in mature sieve elements was quite variable.The parietal layer of cytoplasm in matureUlmus sieve elements consists of plasmalemma, endoplasmic reticulum cisternae in two forms (as a complex network closely applied to the plasmalemma and in stacks along the wall), mitochondria, and plastids.     

OBSERVATIONS ON P-PROTEIN IN DICOTYLEDONS. SUBSTRUCTURAL AND DEVELOPMENTAL FEATURES

The structure and development of P-protein have been studied in sieve elements of hypocotyl tissue of Ecballium elaterium and Cicer arietinum, and in P-protein-producing cells of root apices of Polygonum fagopyrum. Ultrastructural investigations have led us to propose a model for the structure of P-protein tubules. A tubule appears as a Super-Double Helix (“DH1”) which consists of two 6- to 9-nm-diam strands wound round a central lumen, each strand exhibiting a varying-pitched minor double helix (“DH2”). Our observations provide additional insights into the developmental relationships between the different forms of P-protein and support the idea that spiny vesicles participate in P-protein formation. The different types of P-protein bodies found in mature sieve elements of species we have investigated may be regarded as arrays of axially oriented linked “DH1”     

Ultrastructure of Sieve Elements in Secondary Phloem of Dalbegia odorifera During Leaf-Bearing and Leaf-Absent Period

Ultrastructures of sieve elements of secondary phloem of 1–2 year old branchlet of tropical deciduous tree Dalbegia odorifera T. Chen growing on Hainan Island were studied under transmission electron microscope and a comparation was made between the sieve elements in leaf-bearing and leaf-absent period. During the leaf-bearing period, there was a tailed spindleshaped P-protein body in each mature sieve element. The main part of the P-protein body con sisted of a disordered fine fiber mass with two crystalline tails. The sieve elements had horizontal end walls with simple sieve plate. The inner layers of the wall near the sieve plate appeared intumescent, protruding into the sieve element lumen. During the leaf-absent period, a functional phloem remained about the same thickness as that during the leaf-bearing period. The sieve elements in the leaf-absent period contained normal protoplasts and the P-protein and the sieve plate pores had the same structures as those during the leaf-bearing period. More starch grains and vesicles were found in sieve elements in the leaf-absent period.     

TUBULAR AND FIBRILLAR COMPONENTS OF MATURE AND DIFFERENTIATING SIEVE ELEMENTS

An ontogenetic study of the sieve element protoplast of Nicotiana tabacum L. by light and electron microscopy has shown that the P-protein component (slime) arises as small groups of tubules in the cytoplasm. These subsequently enlarge to form comparatively large compact masses of 231 ± 2.5 (SE)A (n = 121) tubules, the P-protein bodies. During subsequent differentiation of the sieve element, the P-protein body disaggregates and the tubules become dispersed throughout the cell. This disaggregation occurs at about the same stage of differentiation of the sieve elements as the breakdown of the tonoplast and nucleus. Later, the tubules of P-protein are reorganized into smaller striated 149 ± 4.5 (SE)A (n = 43) fibrils which are characteristic of the mature sieve elements. The tubular P-protein component has been designated P1-protein and the striated fibrillar component P2-protein. In fixed material, the sieve-plate pores of mature sieve elements are filled with proteinaceous material which frays out into the cytoplasm as striated fibrils of P2-protein. Our observations are compatible with the view that the contents of contiguous mature sieve elements, including the P-protein, are continuous through the sieve-plate pores and that fixing solutions denature the proteins in the pores. They are converted into the electron-opaque material filling the pores.     

Development of P-protein in sieve elements ofMimosa pudica

Summary The P-protein in sieve elements of leaves ofMimosa pudica L. is first discernible as fine fibrous material which forms homogeneous aggregates. Ribosomes, rough endoplasmic reticulum, and dictyosomes with associated vesicles occur in the cytoplasm surrounding the aggregates. The plastids and mitochondria are in a parietal position in the parts of the cell where the nascent P-protein accumulates. In a later stage, the fibrillar material is organized into a three-dimensional system of five- and six-sided elongated compartments. The corners of the compartments appear solid at first, then they become electron lucent in the center and assume tubular form. Aggregates of mature P-protein tubules usually occur near the compartmentalized system. Tubules in pentagonal or hexagonal arrangements may be present in the aggregates and may be partly interconnected. The conclusion was drawn that the P-protein tubules are assembled at the corners of compartments within a continuous orderly system. The fully formed tubules occur first in aggregates, the P-protein bodies. Later the aggregates become loose and partly dispersed. Many of the dispersed tubules assume a loose, extended, helical form characteristic of P-protein in older sieve elements.This work was supported in part by National Science Foundation grant GB-5506. I am also grateful to MissHatsume Kosakai and Mr.Robert H.Gill for technical assistance.     

ULTRASTRUCTURE OF THE NACREOUS LEPTOIDS (SIEVE ELEMENTS) IN THE POLYTRICHACEOUS MOSS ATRICHUM UNDULATUM

The physiological phloem equivalents, leptoids, of the polytrichaceous moss Atrichum undulatum appear to be similar to the nacreous sieve elements that occur in many higher plants. These leptoids are elongated cells with nacreous thickenings on their radial and tangential walls. Their oblique end walls, which lack such thickenings, are traversed by numerous pores through which the plasmalemma, endoplasmic reticulum, and cytoplasm are continuous between adjacent leptoids of a longitudinal file. These end walls closely resemble the simple sieve areas of the sieve elements found in Polypodium vulgare. The leptoid sieve pores have a median expanded area and frequently are occluded by small amorphous protein plugs at each end. Also, callose was observed as electron-luscent areas both on the faces of the end walls and as a thin cylinder surrounding the lateral area of each pore. Amorphous and granular cytoplasmic contents of the leptoids appear to be morphologically similar to the slime (P-protein) found in the sieve-tube elements of many angiosperms. Differentiating leptoids are characterized by the formation of numerous membrane-bound protein bodies in close association with polysomes and endoplasmic reticulum. As the leptoid matures, the contents of the protein bodies become dispersed in the cytoplasm. Ultrastructurally and ontogenetically the leptoids in the gametophores of A. undulatum appear almost identical to the sieve elements of P. vulgare and therefore should be considered sieve elements rather than phloem-like equivalents.     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.     

The ultrastructure and development of tubular and crystalline P-protein in the sieve elements of certain papilionaceous legumes

Summary The ultrastructure of the primary sieve elements of several papilionaceous legumes was studied using hypocotyl and young internode segments fixed in glutaraldehyde followed by osmium tetroxide. In particular, the study sought to determine whether the crystalline flagellar inclusions characteristic of these species are developmentally related to the P-protein bodies present in the phloem of these and other legumes and of angiosperms generally. The crystalline inclusions consist of a central body terminated at one or both ends by a gradually tapering tail. The central body is usually spindle shaped in longitudinal section and square in cross section. In all species examined, the inclusion is first seen as a small, thin crystal in the cytoplasm of young sieve elements. The crystal enlarges and acquires tails as the sieve element develops. In certain species, exemplified byDesmodium canadense, numerous tubules are formed in the cytoplasm near the crystal and appear to be concerned in its growth. The observations on the structure and interactions of these two components, tubules and crystalline inclusions, suggest that both represent forms of P-protein: the tubules are continuous with the crystal and are striated like the crystal near the tubule-crystal junction, suggesting that they are adding onto the crystal body; the tubules closely resemble the P-protein tubules described in the literature in that they measure 157 Å in diameter, accumulate in spindle-shaped bundles, and disperse into striated fibrils late in the ontogeny of the sieve element; and finally, the crystal also disperses into fine filaments. The crystalline inclusion therefore probably represents still another aggregation state of P-protein, one that is characteristic of papilionaceous legumes. The different stages of crystal aggregation and the diverse forms of P-protein now known are discussed briefly in relation to the control of macromolecular assembly and subunit packing.     

P PROTEIN IN THE PHLOEM OF CUCURBITA : II. The P Protein of Mature Sieve Elements

During maturation of sieve elements in Cucurbita maxima Duchesne, the P-protein bodies (slime bodies) usually disperse in the tonoplast-free cell. In some sieve elements the P-protein bodies fail to disperse. The occurrence of dispersal or nondispersal of P-protein bodies can be related to the position of the sieve elements in the stem or petiole. In the sieve elements within the vascular bundle the bodies normally disperse; in the extrafascicular sieve elements the bodies often fail to disperse. Extrafascicular sieve elements showing partial dispersal also occur. The appearance of the sieve plate in fixed material is related to the degree of dispersal or nondispersal of the P-protein bodies. In sieve elements in which complete dispersal occurs the sieve plate usually has a substantial deposit of callose, and the sieve-plate pores are filled with P protein. In sieve elements containing nondispersing P-protein bodies the sieve plate bears little or no callose, and its pores usually are essentially "open." The dispersed P-protein components may aggregate into loosely organized "strands," which sometimes extend vertically through the cell and continue through the sieve-plate pores; but they may be oriented otherwise in the cell, even transversely.     

Formation and dispersal of crystalline P-protein in sieve elements of soybean (Glycine max L.)

Summary Light microscopic observations dating back to 1892 have established that sieve elements of papilionaceous legumes contain a unique type of slime body. This large, compact crystalline type of P-protein has also been observed in sieve elements in recent electron microscopic investigations but its formation and possible relationship to other P-protein structures have not been examined. The present fine structural study describes its development in hypocotyl tissue of 4-day old seedlings of soybean (Glycine max L.). Preceding the formation of a P-protein body, a young sieve element possesses large numbers of ribosomes, abundant vesiculate ER and numerous dictyosomes surrounded by vesicles. A finely granular material accumulates among these components, then condenses into electron opaque masses. Scattered bundles of tubules appear within these masses, then aggregate, and next align longitudinally in the sieve element. By a further transformation, the tubules are converted into an electron opaque crystalline P-protein body. This body continues to grow by aggregation and transformation of additional tubules, and at maturity may be as long as 15–30 microns. The main body, which is square in cross section, tapers toward the ends and is terminated by sinuous tails. Eventually this crystal disperses into a mass of fine striated fibers that fills the lumen of the mature sieve element. Attention is directed to similarities between the bundles of tubules and previously described extruded nucleoli. Factors possibly involved in the structural variations and transformations described above are also discussed.This work was supported in part by grant no. GB-15246 from the National Science Foundation.     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

Ultrastructure of minor veins inCucurbita pepo leaves

Summary The minor veins ofCucurbita pepo leaves were examined as part of a continuing study of leaf development and phloem transport in this species. The minor veins are bicollateral along their entire length. Mature sieve elements are enucleate and lack ribosomes. There is no tonoplast. The sieve elements, which are joined to each other by sieve plates, contain mitochondria, plastids and endoplasmic reticulum as well as fibrillar and tubular (190–195 diameter) P-protein. Fibrillar P-protein is dispersed in mature abaxial sieve elements but remains aggregated as discrete bodies in mature adaxial sieve elements. In both abaxial and adaxial mature sieve elements tubular P-protein remains undispersed. Sieve pores in abaxial sieve elements are narrow, lined with callose and are filled with P-protein. In adaxial sieve elements they are wide, contain little callose and are unobstructed. The intermediary cells (companion cells) of the abaxial phloem are large and dwarf the diminutive sieve elements. Intermediary cells are densely filled with ribosomes and contain numerous small vacuoles and many mitochondria which lie close to the plasmalemma. An unusually large number of plasmodesmata traverse the common wall between intermediary cells and bundle sheath cells suggesting that the pathway for the transport of photosynthate from the mesophyll to the sieve elements is at least partially symplastic. Adaxial companion cells are of approximately the same diameter as the adaxial sieve elements. They are densely packed with ribosomes and have a large central vacuole. They are not conspicuously connected by plasmodesmata to the bundle sheath.     

STRUCTURE AND DEVELOPMENT OF THE SIEVE ELEMENT IN THE STEM OF LYCOPODIUM LUCIDULUM

Stem tissue of Lycopodium lucidulum Michx. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. Although their protoplasts contain similar components, immature sieve elements can be distinguished from parenchymatous elements of the phloem at an early stage by their thick walls and correspondingly high population of dictyosomes and dictyosome vesicles. Late in maturation the sieve-element walls undergo a reduction in thickness, apparently due to an “erosion” or hydrolysis of wall material. At maturity, the plasmalemma-lined sieve elements contain plastids with a system of much convoluted inner membranes, mitochondria, and remnants of nuclei. Although the endoplasmic reticulum (ER) in most mature sieve elements was vesiculate, in the better preserved ones the ER formed a tubular network closely appressed to the plasmalemma. The sieve elements lack refractive spherules and P-protein. The protoplasts of contiguous sieve elements are connected with one another by pores of variable diameter, aggregated in sieve areas. As there is no consistent difference between pore size in end and lateral walls these elements are considered as sieve cells.     

Ultrastructural features of differentiating protophloem sieve elements in adventitious roots of Salix viminalis

The differentiation of the protophloem in 9- to 14-day-old adventitious roots of Salix viminalis was studied. Ultrastructural observations were mainly made on longitudinal serial sections through an uninterrupted file of 32 differentiating sieve elements. The first cell in the file was located about 50 μm from the apical meristem. At an early stage the nucleus was lobed in outline, and in older cells the nucleoplasm became electron lucent. In the first or second cell from the first mature sieve element the nuclear envelope broke open. The nucleoli decreased gradually in size and disappeared finally. From the 9th cell the plastids contained starch and grew somewhat in size. ER increased in amount and began to form stacks in the 20th cell. These stacks moved to a peripheral position. Callose platelets were first observed on the transverse walls in cell 18. Flattened ER-cisternae covered the sieve pore sites. Gradually the middle lamella was dissolved and the callose aggregations formed cylinders around the pores of the sieve plate. Aggregations of tubular P-protein were present from cell 15. P-protein bodies were also present in parenchyma cells adjoining mature sieve elements. The only cell components remaining in mature sieve elements were plastids, mitochondria, stacked ER, the plasmalemma, remnants of other membranes and bodies consisting of P-protein and of an unidentified granular material. The sieve elements had no ontogenetically related companion cells. At a level where both metaphloem and metaxylem had matured the first formed protophloem sieve elements remained intact.     

Monoclonal antibodies against phloem P-protein from plant tissue cultures. II. Taxonomic distribution of cross-reactivity

P-protein, a filamentous protein found in the sieve elements of most angiosperms, is believed to function in the sealing of phloem wound sites. We report here on the use of a highly sensitive immunomicroscopy assay to study the ability of P-protein specific monoclonal antibodies RS21, RS22, and RS23, made against the P-protein from Streptanthus tortuosus (Brassicaceae), to recognize the native P-protein in a number of different plant genera. RS21, RS22, and RS23 all recognized the P-protein in other genera within the Brassicaceae including Arabidopsis and in the closely related family, Capparaceae. RS21 and RS22 also were able to bind to the P-protein in plants more distantly related to S. tortuosus. The labeling of P-protein was also observed in the monocots Iris and Narcissus probed with RS21. No label was seen with members of the Poaceae that are reported to lack P-protein. None of the monoclonal antibodies was able to bind to the P-protein in members of the Cucurbitaceae.     

STRUCTURE AND DEVELOPMENT OF THE SIEVE ELEMENTS IN PSILOTUM NUDUM

Shoot tissue of Psilotum nudum (L.) Griseb. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. Young sieve elements can be distinguished from contiguous parenchyma cells by their distinctive plastids, the presence of refractive spherules, and the overall dense appearance of their protoplast. The refractive spherules apparently originate in the intracisternal spaces of the endoplasmic reticulum (ER). With increasing age the sieve-element wall undergoes a marked increase in thickness. Concomitantly, a marked increase occurs in the production of dictyosome vesicles, many of which can be seen in varying degrees of fusion with the plasmalemma. Other fibril- and vesicle-containing vacuoles also are found in the cytoplasm. In many instances the delimiting membrane of these vacuoles was continuous with the plasmalemma. Vesicles and fibrillar materials similar to those of the vacuoles were found in the younger portions of the wall. At maturity the plasmalemma-lined sieve element contains a parietal network of ER, plastids, mitochondria, and remnants of nuclei. The protoplasts of contiguous sieve elements are connected by solitary pores on lateral walls and pores aggregated into sieve areas on end walls. All pores are lined by the plasmalemma and filled with numerous ER membranes which arise selectively at developing pore sites, independently of the ER elsewhere in the cell. P-protein and callose are lacking at all stages of development.     

Developmental Features of Cell Wall Formation in Sieve Elements of the Grass Aegilops comosa var. thessalica

In differentiating sieve elements of Aegilops comosa var. thessalicadictyosomes are abundant and they produce numerous smooth vesicles.Coated vesicles seem to bud from smooth ones. Since both kindsof vesicles appear both in the cytoplasm and in associationwith the plasmalemma, it is proposed that they move to and fusewith the plasmalemma transferring products for cell wall synthesis.During differentiation sub-plasmalemmal microtubules are initiallyscarce and randomly oriented but soon afterwards they becomenumerous and transversely oriented to the long axis. Cellulosemicrofibrils in the cell wall appear to run parallel to themicrotubules and the latter may regulate microfibril orientation. Root protophloem sieve elements develop wave-like wall thickenings,which are, during development, overlaid by microtubules perpendicularto the long axis. Just after maturation these thickenings progressivelybecome smooth and finally the walls appear uniform in thickness.The wave-like wall thickenings may function as stored wall material,utilized in later stages of development when wall material willbe needed and its synthesis will be impossible because of theabsence of a synthesizing mechanism in the highly degraded protoplastsof mature sieve elements. It is suggested that in this way thethickenings may enable root protophloem sieve elements to growand keep pace with the active clongation of the surroundingcells. Aegilops comosa var. thessalica, sieve elements. cell wall, microtubules, dictyosomes, coated vesicles, wave-like thickenings     

Sieve-plate pores in leaf veins of Hordeum vulgare

Summary The sieve-plate pores of sieve elements in leaf veins of Hordeum vulgare, fixed in glutaraldehyde with postfixation in osmium tetroxide, were lined by the plasmalemma and variable amounts of callose. All pores were filled with endoplasmic reticulum, which was continuous from cell to cell. Mature sieve elements lacked P-protein.