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.     

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.     

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.     

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.     

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.     

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices

Summary A biochemical and cytochemical study has been made of the distribution of -glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of -glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. -glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM -glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. -glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of -glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.     

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.     

P protein in the phloem of Cucurbita. I. The development of P-protein bodies

Light and electron microscopical observations of the cells of the phloem of Cucurbita maxima have shown that two distinct types of P-protein bodies are formed: a larger type which arises as fine fibrils and a smaller type which apparently arises as groups of tubules. The tubules of the smaller type of body measure 242 ± 3.6 (SE) A (n = 48) and appear morphologically identical with the P1-protein tubules of Nicotiana tabacum L. In some of these P1-protein bodies the tubules are arranged in a regular manner with a center-to-center distance of 295 A. The P protein of the larger type of P-protein body is first apparent in the cytoplasm as small aggregates of fine fibrils. This P-protein component has been designated P3 protein. As the P3 protein accumulates it is organized into large bodies. Some of these bodies contain only P3 protein, others a tubular form of protein, and still others a combination of P3 protein and a tubular form. This variability indicates that there is a developmental sequence of the formation of tubules from the P3-protein fibrils. These tubules measure 179 ± 8.2 (SE) A (n = 31) and have been designated P4 protein.     

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.     

Structure and development of sieve cells in the primary phloem ofPinus resinosa

Summary The primary phloem consists mostly of sieve cells. Procambial cells and very young sieve cells contain all the components characteristic of young nucleate cells. Increase in wall thickness, which is relatively limited, constitutes the first indication of sieve-cell differentiation. During the period of wall thickening, the plastids develop starch grains and then fibrillar inclusions. Eventually the internal lamellae of the plastids collapse. The plastids do not form crystalline inclusions. As the sieve cell approaches maturity, an extensive network of smooth, tubular endoplasmic reticulum (ER) appears and then becomes mostly parietal in distribution. At maturity, large aggregates of this ER occur at the sieve areas. These aggregates are interconnected longitudinally by the parietal network of ER. In addition to the ER, the mature, plasmalemma-lined primary sieve cell contains a degenerate nucleus, with intact nuclear envelope, plastids, and mitochondria. Dictyosomes, ribosomes, and vacuoles are lacking. P-protein is not present at any stage of development.This work was supported by U.S. National Science Foundation grants GB 8330 and GB 31417 to R. F.Evert.     

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.     

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.     

Developmental Features of the Primary Phloem in Phaseolus vulgaris L.

Certain developmental features of the primary phloem were examinedin Phaseolus vulgaris L., chiefly by the use of the pulvinusat the base of the petiole. The cells included in the studywere the sieve element, the companion cell, and the tannin cell.In the sieve element, the sieve plate shows the usual sequenceof conversion of plasmodesmatal canals into pores. The endoplasmicreticulum, which appears as flat cisternae associated with ribosomesin younger cells, later becomes in part stacked and in partaligned parallel with the walls as a network. The stacked ERprecedes the anastornosing parietal ER in time of development,but the parietal ER persists longer. Of the two forms of P-proteincharacteristic of a number of Fabaceae, the crystalline bodyappears considerably in advance of the body composed of tubules.Neither form of P-protein disperses completely in the maturecell, although the crystalline protein may spread out into aggregatesof fine fibrils. The companion cells show the typical denseprotoplasts and branched plasrnodesmatal connections with thesieve elements. The vacuome of these cells is dispersed intonumerous small vacuoles, many of which appear to be concernedwith autophagic digestion of protoplasmic material. The tannincells have large vacuoles in which the tannin material is located.The cells form vertical series in which the end walls becomeperforated.     

A Crystalline P-Protein in Gmelina arborea

In a light microscope study of the secondary phloem in Gmelinaarborea (Verbenaceae) many sieve elements were found to possessbar-shaped cytoplasmic inclusions of proteinaceous nature Itis suggested that these inclusions represent a type of crystallineP-protein not reported in the family Verbenaceae before Crystalline P-protein, phloem, sieve element inclusion     

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”     

STRUCTURE AND DEVELOPMENT OF THE SIEVE-ELEMENT PROTOPLAST IN THE HYPOCOTYL OF PINUS RESINOSA

Hypocotyl tissue of Pinus resinosa Ait. was fixed in glutaraldehyde-paraformaldehyde and postfixed in osmium tetroxide for electron microscopy. Although young sieve cells contain all the components characteristic of young, nucleate cells, they can be identified early in their development. Increase in wall thickness occurs early and rapidly. Concurrently, the plastids, which already contain starch granules, form both crystalline and fibrillar inclusions. As the sieve cell approaches maturity, an extensive network of smooth, tubular endoplasmic reticulum (ER), which becomes mostly parietal in distribution, is formed. At maturity, massive aggregates of this ER occur on both sides of sieve areas. These ER aggregates are interconnected with one another longitudinally by the parietal ER. In addition, the mature, plasmalemma-lined sieve cell contains a degenerate nucleus, mitochondria, and intact plastids. Dictyosomes, ribosomes, and vacuolar membranes are lacking. P-protein is not present at any stage of development.     

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.     

Effects of a prolonged treatment with aminoglutethimide on the zona fasciculata of rat adrenal cortex: A morphometric investigation

Summary The effects of a 7-day administration of aminoglutethimide (AG) on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. There was a 70–74% decrease in the concentration of corticosterone in blood, but no conspicuous qualitative changes suggesting cell degeneration occurred. Morphometry showed that AG induced a significant hypertrophy of the zona fasciculata and its parenchymal cells only in normal animals, which was due to an increase in the volume of the mitochondrial compartment and to proliferation of the smooth endoplasmic reticulum. This response to AG was considered to be non-specific and mediated by the enhanced secretion of ACTH following the decrease in the blood level of corticosterone. AG administration significantly increased the volume of the lipid-droplet compartment and the number of intramitochondrial lipid-like inclusions in both groups of animals. These changes were interpreted as the morphological counterpart of the AG-induced block of cholesterol utilization in steroid synthesis.     

Some aspects of sieve-element structure and development inSelaginella kraussiana

Summary The vacuoles of the sieve elements ofSelaginella kraussiana contain a crystalline protein which appears to degenerate in mature cells. Although it occurs in sieve elements we have elected not to call it P-protein because of ontogenetic and possibly functional differences between the two. The nucleus undergoes unique structural changes during development of the sieve element, ultimately being converted to a mass of tubules. The structures referred to by earlier workers as refractive spherules inSelaginella are probably plastids. As the size of the sieve pores in lateral and end walls falls into the same size range, the sieve elements ofSelaginella kraussiana can be considered to be sieve cells.This work was supported by the U.S. National Science Foundation (GB 31417).     

Transformation and development of the flagellar apparatus ofCryptomonas ovata (Cryptophyceae) during cell division

Summary InCryptomonas ovata, long, dorsal flagella are produced which transform during the following cell division into short, ventral flagella. At division there is a reorientation in cell polarity, and the parental basal apparatus, which comprises the basal bodies and associated roots, is distributed to the daughter cells via a complex sequence of events. Flagellar apparatus development includes the transformation of a four-stranded microtubular root into a mature root of different structure and function. Each newly formed basal body nucleates new microtubular roots, but receives a striated fibrous root from a parental basal body. The striated roots are originally produced on the transforming basal body and are transferred to the new basal bodies at each successive division. The development of the asymmetric flagellar apparatus throughout the cell cycle is described.