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.     

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.     

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.     

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 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.     

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     

Fine structure,pattern of division,and course of wound phloem in Coleus blumei

The wound phloem bridges which have developed six days after interrupting an internodal vascular bundle contain wound sieve-elements, companion cells, and phloem parenchyma cells. An analysis of the meristematic activity responding to the wounding clearly demonstrates that three consecutive divisions are prerequisite to the formation of phloem mother-cells. Companion cells are obligatory sister cells of wound sieve-elements, connected to the latter by specific plasmatic strands and provided with a dense protoplast. Six days after wounding most of the wound sieve-elements are still at a nucleate state of development, but already have characteristic P-protein bodies and plastids containing sieve-element starch. Their cytoplasmic differentiation corresponds to the changes recorded during maturation of ordinary sieve elements. Sieve-plate pores penetrate through preexisting parenchyma cell walls, only, and develop from primary pitfield-plasmodesmata. Wound sieve-elements do not connect to preexisting bundle sieve-elements, they open a new tier of young sieve elements produced by cambial activity.     

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”     

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.     

Endoplasmic reticulum derived decorated tubules in the sieve elements ofNymphaea

Summary Bundles of decorated tubules found in the sieve elements ofNymphaea have been studied with the transmission electron microscope. Comparatively straight tubules (100 nm in diameter) arise from the endoplasmic reticulum during early stages of sieveelement development and subsequently associate into bundles of up to 100 tubules that parallel the longitudinal cell axis. From the start of their formation the tubules are structurally distinct from other ER profiles due to their dense decoration with particles. High magnifications reveal an orderly array of the particles (about 24 surround a 100 nm tubule) and suggest a modification of their membrane so that it is no longer dissolvable into a regular three-layered structure. Later during sieve-element ontogeny the decorated tubules get invaginated by smooth ER membranes, thereby squeezing out the intratubular (extracytoplasmic) space. As a result a double mantle is formed that surrounds a plasmatic cylinder. Decorated 100 nm tubules with inner membranes are present in enucleate mature sieve elements ofNymphaea alba andN. tuberosa. Considerably larger tubules (about 200 nm in diameter) were found inN. Candida andN. tetragona and occasionally also inNuphar and Barclaya, two other genera from the same family. The decoration of the tubules and their subsequent invagination by smooth membranes are discussed with respect to the controlled autolysis of sieve elements.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

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.     

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Cytochemical localization of adenosine triphosphatase in the phloem of Pisum sativum and its relation to the function of transfer cells

The cytochemical localization of ATPase in differentiating and mature phloem cells of Pisum sativum L. has been studied using a lead precipitation technique. Phloem transfer cells at early stages of differentiation exhibit strong enzyme activity in the endoplasmic reticulum (ER) and some reaction product is deposited on the vacuolar and plasma membranes. As the phloem transfer cells mature and develop their characteristic wall structures, strong enzyme activity can be observed in association with the plasma membranes and nuclear envelopes. Mature phloem transfer cells with elaborate cell-wall ingrowths show ATPase activity evenly distributed on plasma-membrane surfaces. Differentiating sieve elements show little or no enzyme activity. When sieve elements are fully mature they have reaction product in the parietal and stacked cisternae of the ER. There is no ATPase activity associated with P-protein at any stage of sieve-element differentiation or with the sieve-element plasma membranes. It is suggested that the intensive ATPase activity on the plasma membranes of the transfer cells is evidence for a transport system involved in the active movement of photosynthetic products through these cells.Key to labeling in the figures ER endoplasmic reticulum - P parenchyma cell - PP P-protein - SE sieve element - SPP sieve-plate pore - TC transfer cell     

ULTRASTRUCTURE OF DEVELOPING SIEVE ELEMENTS IN THLASPI ARVENSE L. I. THE IMMATURE STATE

Immature sieve elements of pennycress (Thlaspi arvense, Brassicaceae) were studied with the electron microscope in connection with studies on virus-infected plants. Immature sieve elements contained cytoplasm rich in organelles and other components: endoplasmic reticulum, dictyosomes and associated smooth and coated vesicles, mitochondria, plastids, ribosomes, microtubules, microfilaments, vacuoles, and nuclei that were sometimes lobed. Tubular P-protein (phloem protein) and one to three granular P-protein bodies also were present in the cytoplasm. Coated vesicles may be involved in formation of the granular P-protein body and in some aspect of cell wall development, for in the latter case, they were often seen united with the plasmalemma. The association of coated vesicles with the P-protein body is discussed with reference to proposed concepts of the origin and function of these vesicles.     

Developmental features of protophloem sieve elements in roots of wheat (Triticum aestivum L.)

Summary Protophloem sieve elements (PSEs) in roots of wheat (Triticum aestivum L.) are arranged in single vertical files. The number of PSEs within the files increases by symmetrical divisions, which take place after the completion of asymmetrical (formative) divisions and before the initiation of differentiation. The divisions are preceded by well defined pre-prophase bands (PPB) of microtubules, which surround the nucleus in an equatorial position. In the cytoplasmic region between the nuclear surface and the PPB, perinuclear and endoplasmic microtubules were observed. The perinuclear microtubules are considered as part of the developing spindle, while the endoplasmic ones interlink the perinuclear microtubules with the PPB. Dividing cells do not show any signs of incipient differentiation. The first and most reliable indication of a commencing differentiation is provided by the sieve-element plastids that begin to accumulate dense crystalloid inclusions in the very young PSEs. In mature PSEs plastids contain two kinds of crystalloid inclusions, dense and thin, in a translucent stroma. Depending on the plastid-inclusions criterion it was shown that: (a) the PSEs of a given root do not initiate differentiation at exactly the same stage, (b) the developmental sequence extends to a span of 7–9 actively differentiating PSEs arranged in a single vertical file, and (c) each PSE needs about 16–21 h to pass through the whole developmental sequence. In the last two differentiating PSEs of a file, mitochondria were found to be enveloped by single cisternae of ER. The association is temporary as it is lost in the first PSEs with an autolysed lumen. During differentiation, Golgi bodies were abundant and active in producing vesicles involved in cell wall development. Golgi vesicles were also found among the microtubules of the PPB, but no local thickening was observed. Golgi bodies disorganize in the last stages of autolysis and disappear in mature sieve elements.Abbreviations ER endoplasmic reticulum - MSE metaphloem sieve element - PPB pre-prophase band - PSE protophloem sieve element Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

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.     

Mop-top tubules. The ultrastructure of unusual tubular elements associated with two different leaf symptoms of potato mop-top virus infected potato plants

Summary Potato mop-top virus, one of the most commonly occurring viruses in virus tested stocks of seed potatoes in the United Kingdom induces four different haulm symptoms which are both climate and variety dependent. Ultrastructural examination showed that the aucuba symptom recognizable as bright yellow patches on the leaves, and the mop-top symptom characterised by the dwarfed and bunched habit of the plants, both contained in their leaf cells, tufts and clusters of microtubule-like elements, although the other ultrastructural features associated with each symptom were quite different. These mop-top tubules occurred in the cytoplasm, between the cell wall and the plasma membrane, and in the vacuole, and have been demonstrated in every cell type.The mop-top tubules were 18–22 nm wide with a 2.5–3.0 nm thick wall and often branched. No regular substructure could be discerned. Complete virus particles were rarely seen. These mop-top tubules are compared with plant microtubules and P-protein tubules, and the topic of viral inclusions and their relevance to virus classification is discussed.     

Das Phloem der Haustorien vonCuscuta

Summary Haustoria ofCuscuta odorata R. & P. andC. grandiflora H.B.K. show continuous traces of sieve elements, connecting the phloem of the host with that of theCuscuta shoot. The continuity of this haustorial phloem is discernible by callose fluorescence after staining with aniline blue. The fine structural criteria for sieve tubes are analyzed electronmicroscopically, with special respect to sieve pores, P-protein, and a distinct wall-standing smooth surfaced ER. Within the central part of the haustorium sieve tubes are elongated, while the elements abutting the phloem of theCuscuta shoot are nearly isodiametric in shape. Both elements are associated with rather large companion cells, derived from an unequal division.

     

Polyhedral inclusion bodies in cells of nitrosomonas spec

Polyhedral inclusion bodies were observed in cells of a Nitrosomonas species. They were present in growing cells as well as in resting cells. In thin sections their size was about 130 nm in growing cells and about 185 nm in diameter in resting cells. The bodies were commonly located in the nucleoplasm. They appeared to be bounded by a nonunit membrane and had a granular substructure.In thin sections about 70% of the exponentially grown cells and about 20% of the resting cells of the investigated strain showed 1–7 respectively 1–3 inclusion bodies.