CALLOSE SUBSTANCE IN SIEVE ELEMENTS

The secondary phloem of 6 species of woody dicotyledons was examined for the occurrence of callose on the sieve plates of active sieve elements. Fluorescence and bright-field staining methods were used to detect callose. Tissue from the 6 species was killed and fixed in each of 5 solutions. Some tissue of each species was submerged in the killing solutions as quickly as possible, the remainder within 15 min after removal from the tree. In each species, some active sieve elements of the quick-killed tissue gave negative callose reactions. All active sieve elements of the delay-killed tissue gave positive callose reactions. These and other results suggest that the active sieve elements in the secondary phloem of the species studied normally lack callose and that the extent of callose deposition in these cells depended primarily upon the rapidity with which the sieve-element protoplasts were killed after wounding of the phloem. In addition, bright-field observations of sieve plates of large numbers of sieve elements from a seasonal collection of Tilia americana secondary phloem suggest that the active sieve elements normally lack callose during the growing season and that the inactive sieve elements normally possess it (dormancy callose).     

Ca2+-mediated remote control of reversible sieve tube occlusion in Vicia faba

According to an established concept, injury of the phloem triggers local sieve plate occlusion including callose-mediated constriction and, possibly, protein plugging of the sieve pores. Sieve plate occlusion can also be achieved by distant stimuli, depends on the passage of electropotential waves (EPWs), and is reversible in intact plants. The time-course of the wound response was studied in sieve elements of main veins of intact Vicia faba plants using confocal and multiphoton microscopy. Only 15-45 s after burning a leaf tip, forisomes (giant protein bodies specific for legume sieve tubes) suddenly dispersed, as observed at 3-4 cm from the stimulus site. The dispersion was reversible; the forisomes had fully re-contracted 7-15 min after burning. Meanwhile, callose appeared at the sieve pores in response to the heat shock. Callose production reached a maximum after approximately 20 min and was also reversible; callose degraded over the subsequent 1-2 h. The heat induction of both modes of occlusion coincided with the passage of an EPW visualized by electrophysiology or the potential-sensitive dye RH-414. In contrast to burning, cutting of the leaf tip induced neither an EPW nor callose deposition. The data are consistent with a remote-controlled occlusion of sieve plates depending on the longitudinal propagation of an EPW releasing Ca(2+) into the sieve element lumen. It is hypothesized that forisome plugs and callose constriction are removed once the cytosolic calcium level has returned to the initial level in those sieve tubes.     

FINE STRUCTURE OF PHLOEM CELLS IN RELATION TO TRANSLOCATION IN THE COTTON SEEDLING

When special precautions were taken to permit killing and fixation of sieve elements before they were cut, sieve pores were found to be open. Companion cells were shown to be highly resistant to freezing injury and less plasmolyzable than phloem parenchyma. Plasmodesmata connected parenchyma to parenchyma, parenchyma to companion cells, and companion cells to sieve elements. Their general absence between parenchyma cells and sieve elements points to a specific role of companion cells in sieve tube functioning. EM studies of these cells revealed an ER system which connects the central core of the plasmodesma to the sieve tube. This system may be responsible for active sucrose transport. Callose was always present on sieve plates of mature functioning sieve elements even with the most rapid killing and fixing possible. Extra callose promoted by heating (45 C) an intact stem segment was found to constrict the sieve pores almost completely. Constriction of plasmodesmata in lateral sieve areas also was evident. Fine structure analysis of the blocking mechanism is in accord with evidence obtained by tracer studies.     

CalS7 encodes a callose synthase responsible for callose deposition in the phloem

It has been known for more than a century that sieve plates in the phloem in plants contain callose, a β-1,3-glucan. However, the genes responsible for callose deposition in this subcellular location have not been identified. In this paper we examine callose deposition patterns in T-DNA insertion mutants (cs7) of the Callose Synthase 7 (CalS7) gene. We demonstrated here that the CalS7 gene is expressed specifically in the phloem of vascular tissues. Callose deposition in the phloem, especially in the sieve elements, was greatly reduced in cs7 mutants. Ultrastructural analysis of developing sieve elements revealed that callose failed to accumulate in the plasmodesmata of incipient sieve plates at the early perforation stage of phloem development, resulting in the formation of sieve plates with fewer pores. In wild-type Arabidopsis plants, callose is present as a constituent polysaccharide in the phloem of the stem, and its accumulation can also be induced by wounding. Callose accumulation in both conditions was eliminated in mature sieve plates of cs7 mutants. These results demonstrate that CalS7 is a phloem-specific callose synthase gene, and is responsible for callose deposition in developing sieve elements during phloem formation and in mature phloem induced by wounding. The mutant plants exhibited moderate reduction in seedling height and produced aberrant pollen grains and short siliques with aborted embryos, suggesting that CalS7 also plays a role in plant growth and reproduction.     

Lactic Acid Clearing and Fluorescent Staining for Demonstration of Sieve Tubes

Sieve tubes of the phloem in cleared plant parts can be located by means of a staining reaction specific for callose. The plant part is decolourized in 1:3 glacial acetic acid-95% ethanol and cleared in hot 85% lactic acid at 98-100 C. Callose is not dissolved by this treatment and is then stained with 0.01% analine blue in 0.07 M phosphate buffer, pH 7.5, and observed by fluorescence microscopy. A sieve tube is recognized by the bright yellow fluorescence of the callose on its sieve plates. In most tissues, a natural light yellow fluorescence of the parenchyma cells is evident after the clearing step. This intensifies upon staining with analine blue and tends to make the tissue opaque, but it can be minimized by quick-killing of the tissue before commencing the decolourization. The procedure gives best results when applied to young tissues in which interference from the natural yellow fluorescence of lignified cells such as xylem elements and phloem fibers is minimal. Callose plugs in pollen tubes were also shown in intact, cleared styles.     

Phloem Translocation and Heat-induced Callose Formation in Field-grown Gossypium hirsutum L

Phloem translocation rates in field-grown cotton (Gossypium hirsutum L.) dropped from morning to afternoon and continued to decline toward evening, except that recovery occurred following the hottest afternoon when the maximum temperature was 44 C. Water deficits increased from morning to evening, and severity of deficits generally were proportional to daytime heating. Water stress contributed toward reducing translocation but was not always the governing factor. Callose breakdown appeared to be slower than heat-induced synthesis, and in the evening callose still reflected the influence of high afternoon temperatures. Translocation was considerably reduced when about 50% or more of the hypocotyl sieve plates had large amounts of callose. While heat-induced callose may have reduced translocation because of sieve plate pore constriction, temperatures of 39 to 44 C appeared to inhibit an additional component of translocation as well, possibly in the leaf blade.     

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.     

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.     

Callose in Frankia-Infected Tissue of Datisca glomerata is an Artifact of Specimen Preparation

Abstract: Callose, or β-1,3-glucan, is a plant cell wall polysaccharide that occurs endogenously at distinct sites in a variety of tissues. Callose is also formed in response to stress involving cell membrane perturbation. In sections of chemically-fixed nodule tissue of the actinorhizal host, Datisca glomerata, callose was cytochemically detected within the Frankia -infected cortical cells, as an extensive network of wall material surrounding the microsymbiont, but not in uninfected cortical cells. Callose formation was completely inhibited within the infected cells when 2-deoxy-D-glucose, an inhibitor of callose formation, was included in the tissue fixative. The study concludes that callose deposition in the Datisca nodule infected zone is apparently a stress response to tissue preparation and fixation. However, the rapidity and extent of callose deposition primarily at the symbiotic interface in Frankia -infected cells suggests an unusual predisposition to biosynthesis of β-1,3-glucan in the nodule cortical cells that is related to their interaction with the microsymbiont.     

A Rapid Method for Macerating Phloem

Sieve cells and sieve tube members can be macerated from the phloem of various organs of woody and herbaceous species by au-toclaving the tissue in a mild macerating medium. This treatment does not digest the primary walls or the callose deposits on the sieve areas and sieve plates of the sieve elements. These cells can then be recognized by the fluorescence of their callose after staining with aniline blue. Sometimes adjacent sieve elements fail to separate and one can observe details of their junctures.     

CHLORTETRACYCLINE-INDUCED FORMATION OF WALL APPOSITIONS (CALLOSE PLUGS) IN INTERNODAL CELLS OF NITELLA FLEXILIS (CHARACEAE)1

The formation of chlortetracycline (CTC)-induced wall appositions or plugs in internodal cells of Nitella flexilis (L.) Ag. was studied with light, fluorescence and electron microscopy. These plugs contain callose and pectin. A few minutes after CTC addition plug formation starts by fusion of polysaccharide-containing vesicles (glycosomes) with the plasmalemma. Plug growth is continued by incorporation of glycosome-endoplasmic reticulum (ER) complexes. The cytoplasm near the plug appears dense because of the accumulation of glycosomes and the increased electron density of plasma matrix and organelles. About 1 h after CTC addition plug growth ceases, the cytoplasm recovers to its pretreatment appearance, and a few glycosomes fuse singly with the plug membrane. Crystalline inclusions which consist of hexagonally packed rods are found near the plug. Coated vesicles and coated pits are clearly seen only in very early and late stages of plug formation. Callose is also found in parts of wound plugs produced after mechanical injury. No callose is present in the underlying, highly ordered wound wall. The failure to produce a wound wall beneath CTC-induced plugs appears to be related to the lower number of coated vesicles during plug formation. The possible significance of the partially coated reticulum in plug and wound wall formation is discussed.     

Myosin,microtubules, and microfilaments: co-operation between cytoskeletal components during cambial cell division and secondary vascular differentiation in trees

The immunolocalisation of unconventional myosin VIII ('myosin') in the cells of the secondary vascular tissues of angiosperm (Populus tremula L. x P. tremuloides Michx. and Aesculus hippocastanum L.) and gymnosperm (Pinus pinea L.) trees is described for the first time and related to other cytoskeletal elements, as well as to callose. Both myosin and callose are located at the cell plate in dividing cambial cells, whereas actin microfilaments are found alongside the cell plate; actin and tubulin are both associated with the phragmoplast. Myosin and callose also localise to the plasmodesmata-rich pit fields in the walls of living cells, which are particularly abundant within the common walls between ray cells and between ray cells and axial parenchyma cells in the phloem and xylem. In those xylem ray cells that contact developing vessel elements and tracheids, myosin, tubulin, actin and callose are localised at the periphery of developing contact and cross-field pits; the respective antibodies also highlight the bordered pits between vessels and between tracheids. The aperture of the bordered pits, whose diameter diminishes as the over-arching border of these pits develops, also houses myosin, actin and tubulin. Myosin, actin and callose are also found together around the sieve pores of sieve elements and sieve cells. We suggest that an acto-myosin contractile system (a 'plant muscle') is present at the cell plate, the sieve pores, the plasmodesmata within the walls of long-lived parenchyma cells, and at the apertures of bordered pits during their development.     

Comparative subcellular immunolocation of polypeptides associated with xylan and callose synthases in French bean (Phaseolus vulgaris) during secondary wall formation

The Golgi apparatus of plant cells is thought to be the main site of synthesis of cell wall matrix polysaccharides and the terminal glycosylation of glycoproteins. Much of this evidence still depends on earlier biochemical studies employing subcellular fractionation. However acquiring pure Golgi membranes is still difficult and the question of spatial organisation of glycosyl transferases can be addressed by immunolocation of the enzymes. An antibody to a xylan synthase-associated polypeptide from French bean, the enzyme which synthesises the core polysaccharide for secondary wall xylan, has been raised and shown to inhibit its activity. Xylan is deposited in secondary thickenings and the xylan synthase was only detected in appreciable amounts in developing xylem cells. The location within the Golgi stack was observed throughout the dictyosomes. Some enzyme subunits were also detected in post-Golgi vesicles. A second antibody to a non-catalytic M(r) 65000 subunit of beta 1,3- glucan (callose) synthase was used for a comparative study. Although the bulk of this enzyme has been detected in previous studies at plasmamembrane-wall interfaces in sieve plates and stressed tissue, a Golgi-location can be observed in root tip meristematic cells during cell plate formation. The enzyme was present throughout the stacks. Callose was also immunolocated in a similar manner to xylan in secondary walls and thickenings and in pits in developing xylem. In these cells, the callose synthase was detected at the surface of the growing thickenings and the plasmamembrane within the pits.     

Callose synthesis in higher plants

Callose is a polysaccharide in the form of β-1,3-glucan with some β-1,6-branches and it exists in the cell walls of a wide variety of higher plants. Callose plays important roles during a variety of processes in plant development and/or in response to multiple biotic and abiotic stresses. It is now generally believed that callose is produced by callose synthases and that it is degraded by β-1,3-glucanases. Despite the importance of callose in plants, we have only recently begun to elucidate the molecular mechanism of its synthesis. Molecular and genetic studies in Arabidopsis have identified a set of genes that are involved in the biosynthesis and degradation of callose. In this mini-review, we highlight recent progress in understanding callose biosynthesis and degradation and discuss the future challenges of unraveling the mechanism(s) by which callose synthase operate.Key words: Arabidopsis thaliana, callose, callose synthase, glucan synthase-like, pollen, plasmodesmata, cell plate, stress     

Enhancement of Phloem Exudation from Fraxinus uhdei Wenz. (Evergreen Ash) using Ethylenediaminetetraacetic Acid

Ethylenediaminetetraacetic acid (EDTA) enhanced the exudation of ¹⁴C-labeled assimilates from excised leaflets and whole plant specimens of Fraxinus uhdei Wenz. A 2 millimolar EDTA concentration was found to be most effective in promoting exudation from excised leaflets, while 10 millimolar EDTA was most effective in whole plants experiments. Exudation rate reached a maximum after 24 hours in both experiments. The continuous presence of EDTA throughout the treatment period was required for maximum exudation from excised leaflets. Stachyose, raffinose, verbascose, and sucrose were the principal compounds found to occur in exudate samples. These compounds are typically transported in sieve elements of various Fraxinus species suggesting the exudate was of phloem origin. Electron microscope studies of petiolule sieve plate pores from excised leaflets showed substantially less callose appearing after treatment with EDTA than after H₂O treatment. It is suggested that EDTA enhances phloem exudation by inhibiting or reducing callose formation in sieve plate pores. The exudation enhancement technique described for whole plant specimens is suggested as a useful means of collecting phloem sap and studying translocation in woody plants.     

Callose Deposition and Photoassimilate Export in Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Callose accumulated on sieve plates of phloem of white bean seedlings exposed to excess Co, Ni, or Zn. The callose deposits ranged in thickness and were most pronounced in midribs of unifoliate leaves and their subtending petioles. Lesser callose deposits were found in stems. Although translocation of ¹⁴C was reduced drastically in seedlings exposed to excess metal, no correlation was found between translocated ¹⁴C and the amount of callose in the petioles. It is concluded that the inhibition of phloem translocation in seedlings exposed to excess metal is due to effects other than callose deposition.     

THE FINE STRUCTURE OF SIEVE ELEMENTS OF NEREOCYSTIS LÜTKEANA

The sieve elements of Nereocystis from the base of phylloids contain numerous small vesicles, cytoplasm, ribosomes, and the usual organelles and membrane systems, including nuclei, plastids, mitochondria, dictyosomes, and endoplasmic reticulum. They have a thick secondary wall layer which is deposited along the longitudinal walls and at the sieve plate excluding the sieve pores. The sieve pores range in diameter from 100 to 400 nm and are lined by plasmalemma. The sieve elements from the hollow basal parts of the pneumatocyst show essentially the same features but have larger and fewer vesicles, relatively little cytoplasm, larger sieve pores, 400–900 nm in diameter, and may lack a nucleus. In old sieve elements there are large deposits of callose on the sieve plate and along the longitudinal wall; the vesicles seem to break down, and the protoplast appears necrotic. It is concluded that the trumpet hyphae and sieve tubes are basically the same type of cell, and that the trumpet-shape of the sieve elements is due to their passive stretching during extension growth of the organ in which they occur. There are minor but significant differences among the sieve elements from different regions of the thallus which may reflect possible levels of structural specialization of the sieve elements within the same plant.     

Chitosan-elicited callose synthesis in soybean cells as a ca-dependent process

A new method for the rapid and quantitative fluorometric determination of callose is described. In suspension-cultured cells of Glycine max, synthesis of callose starts within 20 minutes of treatment with chitosan and parallels over hours the accumulation of 1,3-linked glucose in the wall. Poly-l-lysine also elicits callose synthesis. The effect of chitosan is enhanced by Polymyxin B at low concentrations; this antibiotic alone at higher concentrations can also induce callose synthesis. Callose synthesis is immediately stopped when external Ca²⁺ is bound by ethylene glycolbis-(2-aminoethyl ether)-N,N′-tetraacetate or cation exchange beads, and partly recovers upon restoration of 15 micromolar Ca²⁺.     

Differentiation of the Sieve Plate of Cucurbita: A Further View

A re-examination of sieve plate differentiation in Cucurbitamaxima was undertaken in order to determine the relation betweenthe development and dissolution of pore sites and the growthof the permanent part of a sieve plate. Pieces of young petiolesand internodes fixed in glutaraldehyde and post-fixed in osmiumwere used for the study. A pore site first becomes destinguishableby a pair of flat callose collars around a plasmodesma, oneon either side of the sieve plate. The enlargement of the collarsis rapid and the delimitation of the pore sites is more or lesscomplete before any significant thickening has occurred in thesieve plate elsewhere. Later, however, the thickening of therest of the wall overtakes the height of the collars so thateventually the pore sites appear as depressions. The early wallsandwiched between the callose collars remains distinct throughoutpore development. The process of perforation seems to involvea more or less simultaneous disappearance of the callose collarsand the sandwiched layer. Cisternae of endoplasmic reticulumassociated with the pore site seem to be connected with theplasmodesmatal core at all stages of differentiation which addssupport to the view that the endoplasmic reticulum plays anactive role in the process of pore formation.