Sieve-plate pores in tobacco and bean

Summary Tobacco and bean plants were wilted and then fixed as whole plants with formaldehyde-glutaraldehyde for electron microscopy. In some tobacco plants the sieve-plate pores were large, with little callose. Light slime plugs were present, but there was no compaction of P-protein in the pores. Some pores in wilted bean plants were also unplugged. In other plants of both tobacco and bean the sieve-plate pores were plugged. The pores in unwilted control plants of both tobacco and bean were invariably plugged. Tobacco plants were also cut into thin slices and then immediately fixed. In specimens prepared in this way there was little callose in the pores, and many of the pores were not plugged with P-protein. These observations provide additional evidence that sieve-plate pores may be unplugged in vivo.     

The distribution of P-Protein in mature sieve elements of celery

Summary The extent of blocking of sieve-plate pores caused by release of cell turgor was investigated by fixing and processing for electron microscopy a long length of celery (Apium graveolens L.) phloem. Differences in distribution of P-protein within the pores were observed between those cells near the two cut ends, and the central cells.To assess the effect of chemical fixation on the distribution of P-protein, strands of celery phloem (fixed or unfixed, and not treated with cryoprotectants) were frozen in Freon 12 and then freeze-substituted. In sieve elements from unfixed tissue there were a greater number of sieve plates displaying partially open pores.Direct freezing of unprotected phloem tissue in Freon 12 resulted in the formation of ice crystals within the lumen of the sieve elements. Freezing of tissue at rates fast enough to avoid the formation of damaging ice crystals resulted in sieve-plate pores having an unoccluded central channel with a peripheral lining of P-protein. In the lumen of the sieve elements the P-protein filaments occurred as discrete bundles ca. 0.5 m in diameter, and as a parietal layer varying in thickness from 0.1 to 0.5 m.     

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.     

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.     

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.     

LIGHT MICROSCOPE INVESTIGATION OF SIEVE-ELEMENT ONTOGENY AND STRUCTURE IN ULMUS AMERICANA

At maturity the sieve elements of Ulmus americana L. contain a parietal network of very fine strands of slime which is continuous from one sieve element to the next through the sieve-plate pores. Upon injury this parietal network, which is derived from the slime bodies of immature sieve elements, sometimes becomes distorted into longitudinally oriented strands. Some of these strands frequently extend the length of the cells and often are continuous from one sieve element to the next through the sieve-plate pores. At times past such strands have erroneously been interpreted as normal constituents of the mature sieve-element protoplast. Many mature sieve elements of U. americana contain nuclei, which apparently persist for the life of the sieve elements. In addition, some evidence has been found in mature sieve elements for the presence of a membrane which delimits the parietal layer of cytoplasm, including its network of slime strands, from the vacuolar region of the cell.     

Can sieve-element plastids in Panicum maximum (Poaceae) leaves act in the blockage of injured sieve-tube elements?

The ultrastructural features and the plastid changes caused by sample preparation were studied in sieve elements of Panicum maximum leaves. Samples of expanded leaves, taken near the ligule region, were fixed and processed by common light and transmission electron microscopy methods. In mature sieve-tube elements, the protoplast is electron-translucent and plastids are the most frequent organelles. Mitochondria and smooth endoplasmic reticulum segments are also visible and occupy a parietal position within the cell. The plastids are globular and show electron-dense proteinaceous inclusions in the stroma. The protein crystals are predominantly cuneate, but thin crystalloids and amorphous and/or filamentous proteins also occur. The presence of intact plastids plus others in different phases of plastid envelope rupture were interpreted as evidence that this rupture is a normal event in response to injury. This plastid envelope rupture is possibly activated by the release of pressure in the sieve-tube element. After plastid membrane vesiculation, the stroma and the protein crystals are dispersed within the sieve-element ground cytoplasm. The vesicles originating from the plastid envelope move to one cell pole, while protein crystalloids move to the opposite pole and agglomerate in the sieve-plate region. Our findings indicate that these protein crystalloids, which deposit in the sieve plate, may act in sieve-plate pores occlusion, preventing the release of phloem sap, similar to the role of P-protein in dicotyledons.     

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     

Ultrastructural features of well-preserved and injured sieve elements: Minute clamps keep the phloem transport conduits free for mass flow

Summary After chemical fixation following two different preparation procedures, the ultrastructure of mature sieve elements (SEs) was systematically compared in the transport phloem ofVicia faba leaves andLycopersicon esculentum internodes. The SEs in samples obtained by gentle preparation were well preserved, while those in conventionally prepared samples were generally injured. (1) In well-preserved SEs, parietal P-proteins were associated with cisternae of the SE endoplasmic reticulum (ER). Additionally, theV. faba SEs had crystalline P-proteins, and a homogeneous network of filamentous P-proteins occurred in the lumen of theL. esculentum SEs. In injured SEs, all P-proteins were dispersed. (2) In well-preserved SEs, stacked ER cisternae associated with P-proteins lay also on the sieve-plate walls, but passages were kept free in front of the sieve pores. Injured SEs lacked these orderly arranged deposits. Instead, irregular filamentous and membranous materials occluded the sieve pores. (3) In well-preserved SEs, the sieve-pore lumen was free of obstructions, apart from small, lateral coatings of P-proteins. Sieve pores in injured SEs were always occluded. (4) The SE organelles and, in tomato SEs, also the parietal ER located at the longitudinal walls were firmly attached in the SE periphery and stayed in place after injury. The stable parietal attachment is likely exerted by minute, clamplike structures which link the outer membranes of the SE components with one another or to the SE plasma membrane. Single, straight clamps with a length of about 7 nm anchored the SE components directly to the SE plasma membrane. The connections between adjacent SE organelles and/or parietal ER cisternae were mostly twice as long (about 15 nm) and often were branched. Presumably, the long, branched clamps were constituted by the interaction of opposite short clamps. The ultrastructural results are discussed with respect to SE functioning.     

Aspects of sieve element ultrastructure in Primula obconica

Summary At maturity, the enucleate sieve element of Primula obconica is lined with a parietal layer of cytoplasm consisting of plasmalemma, one or more cisterna-like layers of endoplasmic reticulum, numerous mitochondria and plastids, and a membrane which apparently separates these cytoplasmic components from a large central cavity. The central cavity contains numerous longitudinally oriented slime tubules. We believe these tubules normally form strands which run the length of the cell and traverse consecutive cells through the sieve-plate pores. Developmental aspects are discussed.This research has been supported by NSF Grant GB 3193.     

Distribution of P-protein in Mature Sieve Elements of Cucurbita maxima Seedlings Subjected to Prolonged Darkness

A study has been made of the structure of the sieve tubes inthe phloem of seedlings of Cucurbita maxima kept in total darknessfor 2 or 3 days. All cytoplasmic components were found to beparietal in their distribution. The parietal system was closelyapplied to the cell membrane and appeared to be supported bya continuous framework of endoplasmic reticulum (ER) with whichP-protein was intimately associated. The ER-P-protein complexwas highly compact in some sieve elements and loosened to variousdegrees in others. The pores in the sieve plates were eitherunobstructed or occluded by components of the parietal complexin various ways, occlusion not always being accompanied by noticeabledisruption of the parietal system. In visibly undisturbed sievetubes, in which the ER-P-protein complex was in a highly compactstate, occlusion appeared accidental, arbitrary and withoutany alignment of the components present in the pores. It issuggested that the distribution of the cytoplasmic componentsin the parietal position represents a true-to-life conditionof the sieve tube, preserved due to control of the ‘surge’artefact to which transporting sieve tubes are susceptible.However, the organization of sieve tube probably changes withthe state of transport and the highly compact condition of theER-P-protein complex as well as unobstructed or arbitrarilyobstructed sieve plate pores represent a state of ‘rest’or low transport. Cucurbita maxima, P-protein, sieve elements, phloem, seedlings     

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.     

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     

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.     

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.     

ONTOGENY AND STRUCTURE OF THE SECONDARY PHLOEM IN EPHEDRA

The secondary phloem in Ephedra is atypical of the gymnosperms in general and exhibits several angiosperm-like characteristics. The ray system of the conducting phloem consists of parenchymatous, multiseriate rays. The axial system contains parenchyma cells, sieve cells, and unusual albuminous cells reminiscent of the specialized parenchyma cells found in some angiosperms. These cell types may intergrade with each other. P-protein in the developing sieve element appears early in the form of a single, ovoid slime body. Later, smaller slime bodies appear and quickly disperse. In the mature sieve element the single, ovoid slime body is lost, and P-protein is then evident in the form of a parietal cylinder, thread-like strands, amorphose globules, or a slime plug. Necrotic-appearing nuclei are commonly found in mature sieve cells.     

Mature Phloem of Perennial Monocotyledons

Structural aspects of differentiating and mature sieve elements of perennial monocotyledons in general and of palms in particular are presented. As in other angiosperms, an immature sieve element undergoes a profound modification of the protoplast during differentiation. Intact, mature sieve elements lack nuclei, possess a parietal cytoplasm, empty lumen and sieve-plate pores that are free of obstructions. Such a structure is in general agreement with the physiological data obtained from exuding inflorescences of woody monocotyledons. Structural evidence and some tracer experiments indicate that sieve elements in perennial monocotyledons are long-lived and apparently function throughout the lifetime of the organ or the plant.     

STRUCTURE AND DEVELOPMENT OF SIEVE ELEMENTS IN THE LEAF OF ISOETES MURICATA

Leaf tissue of Isoetes muricata Dur. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. The very young sieve elements can be distinguished from contiguous parenchyma cells by their distinctive plastids and the presence of crystalline and fibrillar proteinaceous material in dilated cisternae of the rough ER. During differentiation, the portions of ER enclosing this proteinaceous substance become smooth surfaced and migrate to the cell wall. Along the way they apparently form multivesicular bodies which then fuse with the plasmalemma, discharging their contents to the outside. At maturity, the sieve element contains an elongate nucleus, which consists of dense chromatin material, and remnants of the nuclear envelope. In addition, the mature sieve element is lined by a plasmalemma and a parietal, anastomosing network of smooth ER. Both plastids and mitochondria are present. P-protein is lacking at all stages of development. Tonoplasts are. not discernible in mature sieve elements. The end walls of mature sieve elements contain either plasmodesmata or sieve pores or both, but only plasmodesmata occur in the lateral walls.     

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.     

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.